The beneficial effects of modafinil administration on repeat mild traumatic brain injury (RmTBI) pathology in adolescent male rats are not dependent upon the orexinergic system

The sleep-wake cycle plays an influential role in the development and progression of repeat mild traumatic brain injury (RmTBI)-related pathology. Therefore, we first aimed to manipulate the sleep-wake cycle post-RmTBI using modafinil, a wake-promoting substance used for the treatment of narcolepsy. We hypothesized that modafinil would exacerbate RmTBI-induced deficits. Chronic behavioural analyses were completed along with a 27-plex serum cytokine array, metabolomic and proteomic analyses of cerebrospinal fluid (CSF), as well as immunohistochemical staining in structures important for sleep/wake cycles, to examine orexin, melanin-concentrating hormone, tyrosine hydroxylase


Introduction
Concussion, often referred to as mild traumatic brain injury (mTBI), is a highly debilitating and costly global health issue (Fallesen and Campos, 2020;Izzy et al., 2021).These injuries are commonly associated with falls, contact sports, motor vehicle accidents, and military combat (Voss et al., 2015).Given the perceived notion that these injuries are mild, many individuals fail to seek medical help, return to normal activities, and subsequently place themselves at risk for repeat mTBIs (RmTBI).Epidemiological data suggests that 20 % of adolescents will have sustained a concussion before 10 years of age and experience at least one RmTBI within two years of their first injury (Corrigan et al., 2010;Curry et al., 2019).Both adolescents and young adults are highly susceptible to the cumulative effects associated with RmTBI, with many individuals going on to develop chronic post-concussive symptomologies and structural changes to the brain (Eyolfson et al., 2022;Badea et al., 2018;Angoa-Pérez et al., 2020;Broussard et al., 2018;Christensen et al., 2020a).
Traumatic brain injury (TBI) comprises two related processes: first is the primary mechanical insult and the second is a delayed secondary injury that involves metabolic, chemical, and cellular changes in the CNS (Kumar and Loane, 2012).Although the primary injury triggers the secondary injury to occur, it is the secondary injury that generates the neurological changes that ultimately define the injury-induced deficits (Signorini et al., 1999).Neuroinflammation is a well-known mechanism of the secondary injury and is typically thought to be detrimental to recovery (van Erp et al., 2022).However, research suggests that neuroinflammation has both detrimental and beneficial effects following TBI, and that the nature of its effects likely depend on the phase of recovery (Kumar and Loane, 2012).It has been proposed that the key to developing neuroprotective treatments that generate optimal conditions for repair and regeneration is to promote the beneficial effects of neuroinflammation while minimizing the detrimental/neurotoxic effects (Kumar and Loane, 2012).This is particularly relevant to RmTBI, which has been shown to induce chronic neuroinflammation, neurodegeneration, and persistent deficits, likely due to accumulating secondary injury cascades (Aungst et al., 2014).
In contrast to the periphery, the lymphatic vasculature, which actively removes metabolic waste and excess interstitial fluid (ISF), is believed to be absent from the central nervous system (CNS) (Mendelsohn and Larrick, 2013).Instead, the glymphatic system, named for its dependence on glial water flux and functional resemblance to the lymphatic system, satisfies this requirement for the CNS (Iliff et al., 2012).The glymphatic system uses paravascular tunnel pathways to eliminate macroscopic waste, such as soluble proteins and metabolites, from the CNS (Jessen et al., 2015).Importantly, this highly organized system is responsible for the removal of neurotoxic compounds, including amyloid-β and tau (Nedergaard, 2013).The glymphatic system's waste clearance function is 90 % more efficient during sleep (Xie et al., 2013), which has significant implications for mTBI as sleep disturbances are regularly reported (Piantino et al., 2019).Additionally, sleep disturbances increase neuroinflammation and produce behavioural deficits (Zhu et al., 2012), in response to reduced glymphatic efficiency.Therefore, the relationship between TBI and sleep problems could explain the heightened neurodegenerative risk detected in epidemiological studies of TBI (Barker et al., 2023;Gardner and Yaffe, 2015).
Given this information, the sleep-wake cycle likely plays an influential role in the development and progression of RmTBI-related pathology.Thus, the first aim of this study was to examine the effects of manipulating the sleep-wake cycle on RmTBI outcomes, using a wakepromoting substance following an experimental injury.To address this, we used modafinil (2-[(diphenylmethyl) sulfinyl] acetamide), an FDA-approved medication with wake-promoting properties that is typically prescribed to treat hypersomnolence (otherwise known as excessive sleepiness) in narcolepsy, obstructive sleep apnea syndrome, and shift work sleep disorder (Erman et al., 2007;Mitler et al., 2000;Chapman et al., 2016;Ballon and Feifel, 2006).A previous study suggested that modafinil ameliorates the excessive daytime sleepiness that typically plagues TBI patients (Kaiser et al., 2010); however, its mechanism of action is largely unknown.Additionally, it has been postulated that modafinil could be clinically effective in treating various pathological outcomes associated with concussion, such as depression (Yrondi et al., 2017) and attention-deficit/hyperactivity disorder (ADHD) (Houck et al., 2019;Iaccarino et al., 2018), while posing a lower risk of abuse and adverse effects compared to the stimulants currently used to treat these conditions (Minzenberg and Carter, 2008).Although modafinil has been proposed to act on several systems, including dopamine, NMDA, GABA, adenosine, and serotonin, one of the most promising candidates, given its wake-promoting properties, is the orexinergic system (Ballon and Feifel, 2006).Moreover, given that both severe and mild TBI have been associated with a loss of orexin neurons (also known as hypocretin) (Baumann et al., 2009;Christensen et al., 2023a), this could explain modafinil's ability to ameliorate excessive daytime sleepiness following TBI.Therefore, the second aim of this study was to determine whether the results of modafinil treatment were associated with the action of orexin-A.Orexin-A is an endogenous neuropeptide released exclusively by the lateral hypothalamus (LH) (De Lecea et al., 1998;Sakurai et al., 1998) that plays a role in the promotion and stabilization of wakefulness (Sakurai, 2007;Sakurai et al., 2010).The deficiency of orexin-A has been proposed as the underlying cause of narcolepsy, and, consequently, orexin-A supplementation has been indicated as a therapeutic intervention (Nakamura et al., 2011).Furthermore, given orexin-A's regulatory role in both the sleep-wake cycle and amyloid-β dynamics (Kang et al., 2009), it seems plausible that orexin-A could mediate the relationship between glymphatic dysfunction and mTBI pathophysiology.
Given that both modafinil and orexin-A are known to promote wakefulness, they offer potential benefits for the fatigue and excessive daytime sleepiness often associated with mTBI (Crichton et al., 2020;Ali et al., 2022).However, as the waste clearance abilities of the glymphatic system function most effectively during sleep (Xie et al., 2013) there is a need to understand the balance between sleep and wake promotion for mTBI recovery.Therefore, following exposure to RmTBIs, when the CNS is burdened with increased waste, such as neurotoxic metabolites and pro-inflammatory molecules (Kumar and Loane, 2012;Giza and Hovda, 2014), disrupting sleep by promoting wakefulness may inadvertently hinder recovery and exacerbate neurological outcomes.Consequently, this study sought to investigate the effects of modafinil treatment on RmTBI outcomes and whether these effects were mediated by orexin-A, hypothesizing that both modafinil and orexin-A treatment would exacerbate RmTBI outcomes.

Animals
All experimental procedures were approved by the Alfred Medical Research and Educational Precinct (AMREP) Animal Ethics Committee (E/1933/2019/M).Adult male Sprague Dawley rats (total n = 107; modafinil experiment n = 57, orexin-A experiment n = 50) acquired from the Monash Animal Research Platform (Clayton, Victoria, Australia) were utilized for this study.Throughout the experiment, rats were kept in an animal housing facility, which was temperature controlled (21 • C), maintained on a 12:12 light:dark cycle (lights on 0700), and rats were provided ad libitum access to standard laboratory food and water.Upon arrival, rats were housed in groups (three per cage) and allowed a 5-day housing habituation period before experimental procedures commenced.

Intracerebroventricular (ICV) cannula implantation
At postnatal (P) day 50, rats were weighed and anesthetized with 5 % isoflurane in oxygen (2l/min) via an induction chamber until unresponsive to a toe-pinch.Rats were then transferred to a stereotaxic frame and administered subcutaneous injections of saline (3 ml) and 0.05 mg/ ml buprenorphine (0.05 mg/kg).Throughout the surgery anaesthesia was maintained with 2 % isoflurane in oxygen (1l/min) delivered via nose cone.
A 22-gauge stainless steel guide cannula (62,005, RWD, China) was inserted into the right lateral ventricle using the coordinates in relation to bregma: − 0.2 mm AP, 1.6 mm ML, − 4.0 mm DV.Four stainless steel screws and dental acrylic cement were utilized to anchor the guide cannula to the skull.To maintain the patency of the guide cannula, a stainless-steel dummy cannula (62,106, RWD, China) of the same length was inserted into the guide cannula.The incision was sutured around the intracerebroventricular cannula (ICV) and dental acrylic cement headcap using 4/0 absorbable sutures (C0022204, Zebravet, Australia).

RmTBI induction
Rats were randomly assigned to either the RmTBI or the sham group, with each cage housing rats from both groups.Building upon evidence from our previous studies (Christensen et al., 2023b), the RmTBI group received 5 mTBIs, each spaced 48 h apart, with the first injury occurring 10 days after the ICV cannula implantation surgery (P60).This injury regimen and inter-injury interval has been shown (Christensen et al., 2023b) to induce cumulative neuropathological and behavioural effects while allowing sufficient recovery time for the animals.
The mTBIs were induced using the lateral impact device, which has been described previously in (Mychasiuk et al., 2016;Christensen et al., 2020b).In brief, both RmTBI and sham rats underwent isoflurane anesthetization via an induction chamber.Rats were then placed in a prone position with their temporal lobe placed against a protective headguard.Subsequently, sham rats were removed without injury while the RmTBI rats underwent injury induction, which involved a 50 g weight being propelled (via pneumatic pressure) toward the protective headguard in direct contact with the temporal lobe of the rat at an average speed of 9.22 ± 0.33 m/s.After either the injury or sham insult, rats were placed in a supine position in a clean cage for recovery wherein time-to-right was recorded.

Modafinil, orexin-A, and vehicle microinjections
Five days following the last mTBI, the ICV microinjections commenced.The ICV microinjections occurred daily for the first 4 days whereafter the dosing schedule was shifted to every other day for a total of 15 microinjections for each rat throughout the study.This allowed the drug concentrations to reach substantial levels while reducing the negative side effects associated with chronic ICV microinjections.The timing of ICV microinjections was kept consistent throughout the study, with the microinjections occurring at 8:00 AM.
A 27-gauge stainless steel internal cannula (62,201, RWD, China), which protruded 1 mm beyond the end of the guide cannula, connected to a 25 μl Hamilton syringe via PE-50 tubing (C313CT, P1 Technologies, USA) was used to administer microinjections.However, it is important to note that each drug and vehicle was allocated its own microinjection setup (i.e., internal cannula, Hamilton syringe, and PE-50 tubing) to avoid contamination.
Modafinil (Axon Medchem, Netherlands) was dissolved in 50 % DMSO in aCSF at a concentration of 10 μg/μl.To control for the differing solvents utilized, the vehicle group was randomized to receive either aCSF or 50 % DMSO in aCSF for the course of the study.All drugs and vehicles were adjusted to a pH of 7.4.5 μl of modafinil, orexin-A, or vehicle was injected at a rate of 2 μl/min whereafter the internal cannula was left in place for an additional 3 min to ensure diffusion.

Behavioural testing
An acute behavioural test battery consisting of three behavioural tests (time-to-right, rotarod, and elevated plus maze (EPM)) was conducted to verify injury induction and consistent outcomes across groups prior to drug administration.This behavioural testing was initiated one day following the final injury (P69) and concluded on P71, thus taking place prior to initiation of drug administration.A chronic behavioural test battery, consisting of the rotarod, elevated plus maze (EPM), and hot-cold plate, was initiated 11 days following the final injury (P80) / after 4 ICV injections and concluded on P99.The animals continued to receive ICV injections every other day throughout this testing period.Therefore, the purpose of this chronic behavioural test battery was to examine both the chronic effects of RmTBI and the consequences of modafinil and orexin-A administration on these outcomes.Rotarod was used to assess balance and motor function.Training for rotarod took place one day prior to the first mTBI (P59) and testing occurred one day following the final injury (P69).During training, rats were required to undergo trials, spaced 10 mins apart, until they were able to remain on the rotating rod for 30 s without intervention.Rotarod testing consisted of three scored trials with the device set to increase from 4 to 40 rpm over 5 min.Both the time and speed the rat achieved immediately prior to falling was recorded.The EPM task, which was conducted on P71 and P94, was used to assess anxiety-like behaviour.This task involves recording the movement of the animal during a 5-min trial where it is able to freely explore a raised maze consisting of two open arms and two closed arms.The number of entries and amount of time spent in the open and closed arms was recorded.Finally, hot-cold plate was used as a measure of thermal nociceptive sensitivity.For two days (P95 and P96) prior to hot-cold plate testing, animals were habituated to the apparatus for 2 min.On testing day (P97), animals underwent hot plate testing first, wherein the apparatus base plate was set to 52 • C. Animals were returned to their home cage for an hour, before undergoing cold plate testing, in which the apparatus base plate was set to 2 • C. For both hot and cold plate testing, animals were monitored for signs of pain and the time to reaction was recorded.
The timing of all behavioural testing was kept consistent between cohorts, with ICV injections occurring on the same schedule in relation to behavioural testing for each cohort.Further, testing was consistently completed by a researcher blinded to the experimental paradigm, between 08:00 and 15:00 throughout the study.See Fig. 1 for an illustrative experimental timeline.

Modafinil and orexin-A ELISAs
At the end of the experiment, a subset of animals received an ICV injection of their assigned treatment (modafinil or orexin-A), and their blood was collected in serum separator tubes via cardiac puncture ~15-20 min later.Blood was left to clot for 20 min at room temperature before being centrifuged at 1500 g and 4 • C for 20 min.Following this, serum was aliquoted and stored at − 80 • C. With these samples, modafinil (181,210, Neogen) and orexin-A (S-1374, BMA Biomedicals) ELISAs were conducted to confirm that the ICV injections elevated the systemic levels of the respective substances.Both ELISAs were conducted in accordance with the manufacturer's protocols.All standards, positive, and negative controls, as well as samples were run in duplicate on each 96-well plate.Results of each ELISA were measured with a FLUOstar Omega microplate reader.

Cytokine panel
For the animals that did not receive an ICV injection prior to euthanasia, blood and serum were collected and stored using the same methods outlined above.A subset of the serum samples were analysed using a Luminex Service: Milliplex Rat 27plex cytokine/chemokine assay conducted by Crux Biolabs (Melbourne, VIC), which was completed in accordance with the manufacturer's instructions.In brief, samples were thawed at 4 • C prior to centrifugation at 10000 g for 10 min.Following this, samples were diluted 1:2 and run in duplicate on a Rat Cytokine/Chemokine Magnetic Bead Panel.

Tissue preparation and immunohistochemical processing
Animals were euthanized on P99 (i.e., 31 days post-final injury).Animals were intraperitoneally injected with pentobarbitone prior to transcardial perfusion with phosphate-buffered saline (PBS) followed by 4 % paraformaldehyde (PFA) in PBS.Extracted whole brains were then stored in 4 % PFA in PBS at 4 • C for 24 h.Following this, brains were cryopreserved for 7 days in 20 % sucrose solution.After freezing in optimal cutting temperature (OCT) compound, 20-μm coronal sections were generated from a subset of the brains and mounted on slide with Trajan microscope slides (P72611 Series 2 Adhesive, Trajan Scientific Australia).
To investigate the effects of RmTBI and modafinil or orexin-A treatment on the cell density and connectivity of some of the major neurocircuitry involved in sleep-wake regulation, we examined the LH, basal forebrain (BF), and locus coeruleus (LC) in our immunohistochemical analysis.The LH contains neuronal cell bodies that produce and secrete orexin-A, which is thought to promote and stabilize arousal by exerting excitatory tone on other arousal areas and indirectly inhibiting sleep-promoting regions (Sakurai et al., 2010;De Luca et al., 2022).Two of the main arousal areas that orexinergic fibres project to are the BF and the LC.Orexin-A excites both the cholinergic neurons of the BF and noradrenergic neurons of the LC, which induces the release of acetylcholine and norepinephrine, respectively (Brown et al., 2012).Acetylcholine release from the cortically-projecting cholinergic neuron of the BF is associated with cortical activation and is therefore highest during wakefulness and REM sleep (Brown et al., 2012;Villano et al., 2017).Norepinephrine neurons fire most rapidly during wakefulness, with norepinephrine markedly exciting other arousal centres while inhibiting sleep-promoting areas (Brown et al., 2012).Therefore, the BF and LC play important roles in mediating the arousal effects of the orexinergic system.The LH also contains neuronal cell bodies that produce and secrete melanin-concentrating hormone (MCH), which has been found to facilitate slow wave sleep and REM sleep by inhibiting arousal systems (Monti et al., 2013), thereby essentially opposing the actions of orexin-A.Thus, we sought to determine whether these experimental manipulations affected the orexinergic system, its projections to other important arousal areas, and the sleep-promoting MCH neuronal cell bodies.

Image acquisition and analysis
The 20-μm tissue sections were imaged on a Leica Thunder motorised widefield microscope (Germany), equipped with an LED 3 light source and a sCMOS K5 camera.A HC PL Apo SC2 20×/0.75Imm UV objective was used to image the left and right LH, BF, and LC.The fluorescent channels were collected through: ], [excitation 480-500, emission 515-547 (orexin)] and [excitation 630-650, emission 660-750 (ChAT and tyrosine hydroxylase)].Images were post-processed with the 'Thunder Large Volume Computational Clearing' within the Leica LASx software.
For the LH, orexin and MCH cell bodies were quantified manually in Fiji (Image J) software, with the left and right hemispheres analysed independently.Cell Profiler (Stirling et al., 2021) was used to quantify the interaction between main cell bodies in the BF and LC (ChAT and tyrosine hydroxylase respectively) and the orexin fibres.Cells were identified by minimum-cross entropy global thresholding and object shape.Cell Profiler's 'Relate Objects' function provided a count of all identified ChAT and tyrosine hydroxylase cells that overlapped orexin fibres, within the sampled regions.
For each region of interest, 3-4 sections per brain, and 5 brains per group were analysed.Cell counts were averaged across the 3-4 sections per brain, giving a left hemisphere average and right hemisphere average for each brain.The same was done for co-localization counts.All samples were imaged and analysed by a researcher blinding to the experimental conditions.

Cresyl violet staining
To confirm cannula placement, 20-μm coronal slices mounted on slide from the coordinates of implantation were stained with cresyl violet acetate (0.25 % w/v).Following a 20 min incubation in cresyl violet, slides were submerged in a series of descending concentrations of ethanol.Finally, slides were cleared in xylene and coverslipped.Once dry, slides were imaged on a Leica Aperio AT Turbo Brightfield slide scanner (Monash Histology Platform, Monash University, Australia).See Supplemental Fig. 1 for representative image.

Proteomic analyses
CSF collected at euthanasia samples were diluted in SDS lysis buffer (5 % w/v sodium dodecyl sulphate, 100 mM HEPES, pH 8.1), heated at 95 • C for 10 min and then probe-sonicated before measuring the protein concentration using the BCA method.The samples were denatured and alkylated by adding TCEP (Tris(2-carboxyethyl) phosphine hydrochloride) and CAA (2-Chloroacetamide) to a final concentration of 10 mM and 40 mM, respectively, and the mixture was incubated at 55 • C for 15 min.Sequencing grade trypsin was added at an enzyme to protein ratio of 1:50 and incubated overnight at 37 • C after the proteins were trapped using S-Trap mini columns (Profiti).Tryptic peptides were sequentially eluted from the columns using (i) 50 mM TEAB, (ii) 0.2 % formic acid and (iii) 50 % acetonitrile, 0.2 % formic acid.The fractions were pooled and concentrated in a vacuum concentrator prior to MS analysis.
Using a Dionex UltiMate 3000 RSLCnano system equipped with a Dionex UltiMate 3000 RS autosampler, an Acclaim PepMap RSLC analytical column (75 μm × 50 cm, nanoViper, C18, 2 μm, 100 Å; Thermo Scientific) and an Acclaim PepMap 100 trap column (100 μm × 2 cm, nanoViper, C18, 5 μm, 100 Å; Thermo Scientific), the tryptic peptides were separated by increasing concentrations of 80 % acetonitrile (ACN)/0.1 % formic acid at a flow of 250 nl/min for 158 min and analysed with an Orbitrap Fusion Tribrid mass spectrometer (Thermo-Fisher Scientific).The instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition.Each survey full scan (375-1800 m/z) was acquired with a resolution of 120,000 (at 200 m/z), a normalised AGC (automatic gain control) target of 250 %, and a maximum injection time of 54 ms.Dynamic exclusion was set to 15 s.The most intense multiply charged ions (z ≥ 2) within a 2 s window following each survey scan were sequentially isolated and fragmented in the collision cell by higherenergy collisional dissociation (HCD) using a Collision Energy of 32 %.All ms2 scnas were acquired in the Obritrap with a fixed injection time of 54 ms, 30,000 resolution and a normalised AGC target of 400 %.The raw data files were analysed with the MaxQuant software suite v1.6.5.0 (Cox and Mann, 2008) to obtain protein identifications and their respective label-free quantification (LFQ) values using standard parameters.These data were further analysed with LFQ-Analyst (Shah et al., 2020).

Metabolomic analyses
CSF samples collected at euthanasia were thawed on ice before four volumes of ice-cold extraction solvent (1:3 chloroform:methanol v/v, internal standards: 2 μM CHAPS, CAPS, PIPES and TRIS) was added.The samples were mixed for 30 min at 4 • C, centrifuged (20,000 ×g, 4 • C, 15 min) and the supernatant was transferred to MS vials for untargeted metabolomic analysis using a Dionex RSLC3000 UHPLC coupled to a Q-Exactive Plus Orbitrap MS (Thermo Fisher Scientific).Samples were analysed by hydrophilic interaction liquid (HILIC) chromatography utilizing a ZIC-p(HILIC) column 5 μm 150 × 4.6 mm with a 20 × 2.1 mm ZIC-pHILIC guard column (Merck Millipore, Australia).A gradient elution of 20 mM ammonium carbonate (A) and acetonitrile (B) was used at 25 • C at a flow rate of 300 μL/min as follows: At t = 0 min, 20 %A increasing to 50 % at t = 15 min, then increasing to 95 % A at t = 18 min before remaining constant until t = 21 min, then returning to 20 % A at t = 24 min and remaining constant until t = 32 min MS scans were performed at a resolution of 70,000 operating in rapid switching positive (4 kV) and negative (− 3.5 kV) mode electrospray ionization (capillary temperature 300 • C; sheath gas flow rate 50; auxiliary gas flow rate 20; sweep gas 2; probe temp 120 • C).Accurate metabolite identification was facilitated by analysing a standard library before sample acquisition (Creek et al., 2011).Acquired LC-MS data were processed using the open source software IDEOM, which initially used msConvert (ProteoWizard) (Chambers et al., 2012) to convert raw LC-MS files to mzXML format, and XCMS to select peaks to convert to .peakMLfiles.Mzmatch was subsequently used for sample alignment and filtering (Scheltema et al., 2011), and IDEOM was utilized for further data pre-processing, organisation, and quality evaluation (Chambers et al., 2012).

MetaboAnalyst
MetaboAnalyst 6.0 (https://metaboanalyst.ca/) was used for further downstream analysis of metabolites, using the statistical analysis [one factor] module.These analyses removed animal 39, belonging to the vehicle RmTBI group, as this sample was contaminated.Data analysed was normalised by the median, log transformed, and pareto scaled.Significantly altered metabolites for selected pairwise comparisons are listed in Supplementary Tables 1-3.

Pathway analysis
Prior to analysis using the pathway analysis module of Metab-oAnalyst 6.0, duplicate IDs were filtered retaining the feature having the largest mean intensity.Due to this, and because some IDs did not have corresponding KEGG codes, 307 metabolites were used in the final processing.This data was then sorted into three groups; vehicle sham against vehicle RmTBI, vehicle sham against modafinil RmTBI, and vehicle sham against orexin-A RmTBI.This was then entered into MetaboAnalyst 6.0 (https://metaboanalyst.ca/), and into the module pathway analysis.Metabolites were identified by the program through their KEGG IDs, data analysed was normalised by the median, log transformed, and pareto scaled, and the pathway library was set to Rattus norvegicus (KEGG).For pathways that were deemed significant (p < .1),KEGG codes were manually confirmed.Raw data for significantly altered pathways when examining RmTBI in vehicle, modafinil, and orexin-A treated animals can be found in Supplementary Tables 4-6, respectively.

Statistical analyses
Data was analysed using SPSS (27.0 for Mac) and two-way ANOVAs, with injury and treatment as factors, where p values of <0.05 were considered statistically significant.When appropriate, pairwise Bonferroni post-hoc analyses were conducted.Graphs were created using GraphPad/Prism 9 software, with all graphs displaying mean ± standard error of the mean (SEM).All raw data will be available at the Open Source Framework repository upon publication.

Modafinil administration
Prior to commencing drug administration, time-to-right, rotarod, and EPM, were used to confirm injury induction.Results from the twoway ANOVAs confirmed that rats in the RmTBI groups took significantly longer to regain consciousness as measured by time-to-right (main effect of injury, F (1,56) = 15.076,p < .001;Fig. 2A), spent less time on the rotarod (main effect of injury, F (1,56) = 6.654, p = .013;Fig. 2B), and spent less time in the open arms of the EPM (main effect of injury, F (1,56) = 15.499,p < .001;2C).Following chronic administration of modafinil, a battery of behavioural tests was used to assess its effects on recovery.At the chronic timepoint, all animals had fully recovered on the rotarod task (no main effects, p > .05;Fig. 2D).Animals in the RmTBI group that received administration of the vehicle continued to spend less time in the open arms of the EPM.Although the RmTBI animals in the modafinil group significantly differed from the RmTBI animals in the vehicle group, there were no differences between RmTBI and sham animals in the modafinil treated groups (main effect of injury, F (1,49) = 5.102, p = .029;injury by treatment interaction, F (1,49) = 4.092, p = .045;2E).While there were no significant findings for the cold plate (p's > 0.05), there was a significant interaction on the hot plate, whereby RmTBI reduced thermal sensitivity in the vehicle treated groups, but this effect was eliminated in the modafinil treated animals (F (1,53) = 4.422, p = .040;2F).At euthanasia, neither injury nor modafinil treatment had any effect on spleen weight (p's > 0.05), however, modafinil did significantly increase brain weight (main effect of treatment, F (1,53) = 5.606, p = .022;2I).
Prior to euthanasia, a subset of animals received an ICV injection of modafinil and serum was collected 30 min later to confirm that the injections elevated systemic levels of modafinil.The modafinil ELISA was a detection ELISA, and therefore could only determine if modafinil was present or not in the sample.All samples in the modafinil group exhibited positive detection (6/6), whereas the vehicle samples (0/4) did not generate positive detection (data not shown).In addition, a multiplex ELISA was used to examine circulating cytokine levels in the serum.We found a main effect for modafinil treatment in 4 cytokines (IP-10, Il-5, MCP-1, and RANTEs), and a significant main effect of injury in 13/24 cytokines, and no significant interactions.See Table 1 and Fig. 3 for statistical and graphical results, respectively.
Next, we used immunohistochemical staining to examine neuropathological changes.Contrary to our hypotheses, RmTBI and modafinil treatment did not affect the number of orexin-A positive cells in the LH (p's > 0.05), although modafinil treatment did reduce the number of MCH positive cells in both RmTBI and sham animals (main effect of treatment, F (1,39) = 6.076, p = .019).See Fig. 4. Within the BF, the number of ChAT positive cells was reduced in sham animals treated with modafinil, but there was no difference in RmTBI animals treated with vehicle or modafinil (significant injury by treatment interaction, F (1,49) = 4.183, p = .047).When we examined the number of co-localized ChAT cells and orexin-A fibres, we found a main effect of injury, treatment, and a significant interaction; whereby modafinil reduced the degree of co-localization, and RmTBI reduced co-localization in vehicle treated animals (F (1,53) = 7.971, p = .008;F (1,53) = 10.570,p = .003;F (1,53) = 16.555,p < .001,respectively).See Fig. 5.We also investigated the quantity of TH positive cells in the LC and found that modafinil treatment significantly increased the number of cells, particularly in RmTBI animals (main effect of treatment, F (1,53) = 4.588, p = .037).When examining the number of co-localized TH cells and orexin-A fibres, we found a main effect of injury, treatment, and a significant interaction; whereby modafinil reduced the degree of co-localization, and RmTBI significantly reduced the co-localization of TH and orexin-A in vehicle treated animals, but there were no differences between RmTBI and sham animals in the modafinil treated groups (F (1,53) = 4.807, p = .033;F (1,53) = 4.224 p = .045;F (1,37) = 5.526, p = .023,respectively).
Finally, untargeted quantitative proteomic and metabolomic analyses were conducted on CSF that was collected prior to euthanasia.When comparing vehicle treated RmTBI animals to vehicle treated sham animals, no proteins were significantly modified.There were however 29 significantly modified (p < .05)putative metabolites, such as 3-O-Sulfogalactosylceramide (d18:1/24:1).See Fig. 6 for illustrative demonstration of change in the metabolites and Supplementary Table 1 for the complete list.In addition, pathway analyses of the metabolites revealed significant changes in 2 pathways, sphingolipid metabolism and fatty acid elongation.See Supplementary Table 4.When examining the effect of modafinil treatment, one protein, F1M1R0 was observed to be significantly upregulated in the modafinil treated RmTBI group when compared to the sham vehicle group (adjusted p = .0128,log2 fold change, 2.88).Principal component analysis of both the modafinil treated and modafinil RmTBI groups found that the metabolomic profiles largely overlapped between modafinil treated and vehicle groups (Fig. 6A).Metabolomic analyses demonstrated significant modulation of 26 metabolites, which included upregulation of myristoleic acid, see Fig. 6B.In addition, pathway analyses of metabolites demonstrated significant changes in 3 pathways: nicotinate and nicotinamide metabolism, beta-Alanine metabolism, and biosynthesis of unsaturated fatty acids.All significantly regulated metabolites and their pathways can be found in supplementary Tables 2 and 5, respectively.

Orexin-A administration
To determine if the beneficial effects of modafinil treatment were associated with orexin-A, we repeated the study protocol, but administered orexin-A rather than modafinil.Similar to what was described above, prior to commencing drug administration, time-to-right, rotarod, and EPM, were used to confirm injury induction.Results from the twoway ANOVAs confirmed that rats in the RmTBI groups took significantly longer to regain consciousness as measured by time-to-right (main effect of injury, F (1,49) = 41.310,p < .001;Fig. 7A), spent less time on the rotarod (main effect of injury, F (1,49) = 4.564, p = .038;7B), and spent less time in the open arms of the EPM (main effect of injury, F (1,49) = 4.391, p = .042;7C).Following chronic administration of orexin-A, a battery of behavioural tests was used to assess its effects on recovery.At the chronic timepoint, RmTBI animals spent more time on the rotarod (main effect of injury, F (1,46) = 5.694, p = .021;7D) and continued to spend less time in the open arms of the EPM compared to the sham animals (main effect of injury, F (1,46) = 5.694, p = .021;7E); orexin-A did not have an effect on either outcome.There were also no main effects of injury or orexin-A treatment on thermal sensitivity with the hot and cold plate (p's > 0.05).Interestingly, both spleen weight and brain weight were significantly reduced in RmTBI animals with orexin-A failing to modify either outcome (main effect of injury, F (1,47) = 10.057,p = .003;F (1,46) = 3.955, p = .050,respectively; 7H & 7I).
Prior to euthanasia, a subset of animals received an ICV injection of orexin-A or vehicle (n = 9/group) and serum was collected 30 min later to confirm that the injections did elevate the systemic levels of orexin-A.The one-way ANOVA demonstrated a main effect of treatment, F (1,17) = 7.574, p = .014,whereby orexin-A levels were significantly higher in the treated group than the vehicle (45.270 ng/ml ± 5.913 and 22.256 ± 4.751, respectively), data not shown.
Additionally, a multiplex ELISA was used to examine circulating cytokine levels in the serum.We found a main effect for orexin-A treatment for IL-1α, no cytokines that were significantly modified by injury, but 14/24 cytokines exhibited significant injury by treatment interactions.See Fig. 8 and Table 3 for graphical and statistical results, respectively.
Next, we used immunohistochemical staining to examine neuropathological changes.There was a significant injury by treatment interaction for the number of orexin-A positive cells in the LH, whereby orexin-A treatment reduced cell numbers in sham animals, but increased cell numbers in RmTBI animals (significant injury by treatment interaction, F (1,41) = 4.674, p = .037).See Fig. 9. Contrary to what we found with modafinil treatment, neither RmTBI nor orexin-A modified the number of MCH positive cells in the LH (p's > 0.05).When examining the BF, we found that RmTBI reduced the quantity of ChAT positive cells (main effect of injury, F (1,38) = 9.554, p = .004),as well as the degree of co-localization between ChAT and orexin-A (main effect of injury, F (1,34) = 19.014,p < .001).See Fig. 10.Lastly, within the LC, the twoway ANOVA revealed an injury by treatment interaction (F (1,38) = 5.125, p = .028),whereby RmTBI reduced TH positive cells in vehicle treated animals, but there was no difference in RmTBI animals treated with orexin-A.Similar results were identified for the co-localization between TH and orexin-A, where we demonstrate a main effect of treatment, F (1,38) = 4.216, p = .045,as well as a significant injury by treatment interaction, F (1,38) = 10.064,p = .003.See Fig. 10.
Finally, as was completed with the modafinil treated animals, proteomic and metabolomic analyses were conducted on CSF that was collected prior to euthanasia for those treated with orexin-A.When comparing orexin-A treated RmTBI animals to vehicle treated shams there were no significant proteins identified.Similar to modafinil treated groups, principal component analyses of orexin-A treatment to  3 for all data.Finally, pathway analyses of the metabolites revealed significant changes in 22 pathways, including the tyrosine metabolism pathway.See Supplementary Table 6.

Discussion
Given that modafinil promotes wakefulness and would therefore theoretically reduce glymphatic clearance of macroscopic waste post-RmTBI, we hypothesized that chronic administration of modafinil would exacerbate injury-related impairments.Contrary to our hypothesis, modafinil administration did not exacerbate injury-related deficits, but did ameliorate anxiety-related dysfunction in the EPM and thermal hypersensitivity on the hot plate.Although not generally known for its analgesic properties, modafinil has been shown to exert analgesic effects on neuropathic pain via the nitrergic and serotonergic system, which may explain the improvement in nociceptive sensitivity in our study (Ghorbanzadeh et al., 2021).However, the analgesic effects in neuropathic pain, were associated with reduced TNF-α and IL-6 levels; a finding we failed to replicate.We found circulating cytokine levels to be largely unaffected, with only IL-5 and MCP-1 exhibiting modafinilinduced reductions, suggesting that the anti-inflammatory actions of modafinil may vary in response to different injury and dosing paradigms.Additionally, modafinil is proposed to activate the noradrenergic neurons of the LC (Hou et al., 2005a), which plays an important role in pain modulation (Llorca-Torralba et al., 2016), and this may represent another potential pathway that modafinil exerts analgesic effects.Though the mechanisms driving the neuroprotective actions of modafinil are currently unknown, it has been trialled in the treatment of numerous neurological conditions (Minzenberg and Carter, 2008).The neuroprotective actions demonstrated within our study may be linked to modafinil's ability to attenuate oxidative damage and aid in the recovery of neurosecretory coupling mechanisms in neurons following exposure to glutamate excitotoxicity (Antonelli et al., 1998), as glutamate toxicity is common contributor to secondary TBI-induced injury cascades.
Although our laboratory has previously demonstrated that RmTBI results in a loss of orexin cells in the hypothalamus acutely post-RmTBI (Christensen et al., 2023a), this loss appears to recover chronically, as neither RmTBI or modafinil treatment affected orexin cell quantity in this study.However, administration of modafinil did reduce the number of MCH positive cells in the LH and ChAT positive cells in the BF, while preventing the loss of TH positive cells in the LC that was identified in animals exposed to RmTBI who received vehicle treatment.Although modafinil is a relatively dirty drug, mouse knockout lines demonstrate that MCH receptor binding is necessary for modafinil-induced wakefulness (Adamantidis et al., 2008).It is possible that modafinil contributed to apoptosis of MCH positive cells as a compensatory mechanism to offset the heightened activity induced within these arousal systems in response to prolonged modafinil administration (McGinty and Alam, 2013).Interestingly, there are also twice as many MCH neurons, when compared to orexinergic neurons (McGinty and Alam, 2013), suggesting that this differences in cell prevalence may have contributed to our ability to detect cell loss.In the context of RmTBI however, these results also indicate that modafinil is protective against the loss of TH positive cells.MCH neurons are known to be silent during wakefulness and increase firing during sleep, reaching maximal levels during REM sleep (Monti et al., 2013).As such, the loss of MCH neurons suggests a reduction in time spent asleep.Although not quantified, reduced time asleep was qualitatively confirmed with video recordings obtained following modafinil administration.Given what we know about sleep, glymphatic clearance, and TBI-induced neuropathology, the reduction in MCH and sleep quantity further suggests that the neuroprotective effects of modafinil that we identified, act in a manner distinct from its wake-promoting properties.
It is well established that the homeostatic drive to sleep is largely controlled by the BF, which contains excitatory cholinergic and inhibitory GABAergic projections to the ventrolateral preoptic nucleus (VLPO) (Gerrard and Malcolm, 2007).ChAT is responsible for the synthesis of acetylcholine, which is important for arousal, attention, and motivation (Oda, 2002).We found that modafinil reduced ChAT in the BF of sham animals, while both RmTBI and modafinil administration reduced the degree of co-localization between ChAT and orexin-A.Given this, it is plausible that modafinil is driving apoptosis in ChAT positive cells while RmTBI is responsible for damaging the orexin-A fibres, thereby leading to the death of these neurons and their connections.This reduction in ChAT would reduce arousal and excitatory input to VLPO, which may have served as a compensatory mechanism to offset the continuous wake-promoting action of chronic modafinil administration.Moreover, TBI often induces a drive for increased sleep (Rowe et al., 2014), which may result from reduced BF ChAT.However, given that some researchers have suggested that modafinil's neuroprotective actions may be particularly driven by adenosine modulation within the BF (Gerrard and Malcolm, 2007), it is possible that ChAT modulation provides similar protective qualities.
Although RmTBI reduced TH levels in the LC of vehicle treated animals, modafinil administration attenuated this loss and animals in the RmTBI-modafinil group were indistinguishable from vehicle shams.Modafinil's effects on arousal must be mediated by input to the LC (Hou et al., 2005b), as modafinil does not change the firing rate of noradrenergic neurons in the LC (Akaoka et al., 1991) or bind to any cells within this brain structure (Lin et al., 2018).To date, the only CNS components known to bind modafinil are the dopamine transporter (DAT) and the norepinephrine transporter (NET) (Madras et al., 2006) and a small subset of neurons in the BF (Lin et al., 2018), suggesting that modafinil acts upstream of the LC.Interestingly, reduced TH is associated with decreased catecholamine synthesis, in part due to decreased nitric oxide signalling (Lerner et al., 2019).Conversely, improvements within the nitrergic system have previously been linked to the neuroprotective properties of modafinil (Ghorbanzadeh et al., 2021), suggesting that the increase in TH identified in our RmTBI -modafinil group may be providing neuroprotection in an analogous manner.Moreover, given the role of the LC's noradrenergic system in pain modulation (Llorca-Torralba et al., 2016), the finding that modafinil attenuated the loss of TH positive cells in the LC following RmTBI could account for the improvement in nociceptive activity that we observed in this group.Metabolomic analyses of modafinil treated RmTBI animals identified changes in 26 putative metabolites, including upregulation of myristoleic acid.Myristoleic acid is an omega-5 fatty acid synthesized from myristic acid.Of particular importance, omega-5 fatty acids have strong anti-inflammatory and antioxidant properties (Zamora-Lopez et al., 2020).As changes in this metabolite and metabolites of similar composition, i.e., 3-hydroxy-tetradeconic acid, were identified in modafinil treated, but not vehicle treated RmTBI animals, this may have contributed to the neuroprotective actions of modafinil administration.In addition, the subsequent pathway analyses derived from the metabolomic analyses demonstrated changes to many metabolites involved in aspects of the nicotinate and nicotinamide metabolism pathway may be modified in the modafinil RmTBI group when compared to the vehiclesham group.Nicotinate or niacin, also known as vitamin B3, is the precursor to the coenzymes, nicotinamide-adenine dinucleotide (NAD + ) and nicotinamide-adenine dinucleotide phosphate (NADP + ).NAD + is one of the most abundant metabolic intermediaries, being involved in approximately 500 enzymatic reactions.As a result of its significant role in metabolic processes, pathologies that increase bioenergetic stress, such as brain injuries, significantly deplete NAD + levels (Klimova et al., 2018).Prolonged disequilibrium of NAD+ metabolism, as identified here with our model of RmTBI, is a prominent feature of neurodegenerative disorders and accelerated aging (Klimova et al., 2018).It is possible that chronic administration of modafinil reduced the accumulation of metabolic toxicity that typically accompanies successive secondary injury cascades that generally result from repeat brain injuries.
Given that modafinil's antioxidant and neuroprotective effects are not believed to be associated with its wake-promoting effects (Gerrard and Malcolm, 2007), and it is a "dirty drug" stimulating release of numerous neurotransmitters, including histamine, norepinephrine, serotonin, dopamine, and orexin, we aimed to determine if its neuroprotective capacity in RmTBI was associated with the orexinergic system.In an effort to dissociate the neuroprotective properties from sleep modulation and glymphatic function, we used orexin-A, which has higher affinity for the orexin-1 receptor (ORX1).ORX1 contributes minimally to sleep-wake behaviour, as sleep regulation is believed to be driven by activation of OXR2 (Wang et al., 2018).If the neuroprotective capacity of modafinil is acting through the orexinergic system in a manner independent of wake-promotion, the effects of orexin-A treatment post-RmTBI should be comparably beneficial.Like modafinil, orexin-A has been shown to have neuroprotective and analgesic properties, although the mechanisms have also yet to be elucidated (Wang et al., 2018;Kitamura et al., 2010;Askari et al., 2021).It is believed that orexin-A treatment is capable of down-regulating phagocytosis which may be beneficial in some circumstances; however, this process also reduces the degradation of molecules such as amyloid-β and Tau, which may be detrimental for RmTBI and other neurodegenerative conditions (An et al., 2017).
Again, contrary to our hypotheses, and in contrast to the modafinil findings, administration of orexin-A did not ameliorate RmTBI-induced behavioural deficits.However, in line with our original hypothesis that wake-promotion would exacerbate RmTBI-induced deficits, orexin-A treated RmTBI animals exhibited exacerbated motor deficits as measured with the rotarod as well as persistent changes in anxiety-like behaviour.Increased serum levels of orexin-A have been identified in adolescents with diagnosed anxiety-disorders (Akca et al., 2019), and hyperactivity of the orexinergic system has been implicated in the maintenance of high arousal and anxiety (Flores et al., 2015).Our chronic administration of orexin-A may have contributed to the maintenance of RmTBI-induced anxiety.
Despite exacerbation of some behavioural findings, orexin-A administration remediated many of the RmTBI induced changes in cytokine levels (e.g., fractalkine, IFN-Y, IL-1B, IL-2, IL-5, TNF-a); and when administered to sham animals, it reduced circulating levels of inflammatory cytokines.This is consistent with previous literature demonstrating that orexin-A can diminish the production of reactive oxygen species and act as anti-inflammatory neuropeptide.For example, studies employing models of rheumatoid arthritis and LPS-induced endotoxin shock have demonstrated that treatment with orexin-A reduces the secretion of numerous cytokines (i.e., IL-1B, IL-6, IL-8, and TNF-a), reduces neuronal death, and increases overall survival (Sun et al., 2018;Grossberg et al., 2011).It is important to note, that at least at the chronic time point measured within this study, RmTBI may have resulted in suppression of immune system activity.Cytokine levels for RmTBI animals that did not receive modafinil or orexin-A treatment were often lower than sham animals.Similar to chronic stress, where immunosuppression may occur to reduce the risk of a hyperactive autoimmune response (Raberg et al., 1998), the chronic recovery process associated with RmTBIs may induce immune suppression in an attempt to provide similar protection for neuroinflammatory functions.
Orexin-A administration also significantly reduced the quantity of orexin cells in the LH of sham animals.This was expected, as the number of orexin cells would decrease to compensate for the supraphysiological levels of orexin-A that we administered.Interestingly, this was not the case in animals with RmTBI.This group's orexin cell count was equivalent to vehicle treated animals, suggesting that within the context of RmTBI, supraphysiological orexin-A levels did not result in similar compensatory changes.It is possible that the RmTBI pathology reduced the brain's own generation of orexin-A or increased its capacity to process orexin-A through a distinct mechanism.
Interestingly, orexin-A administration had no effect on the number of MCH cells in the LH.Within the LH, orexins activate MCH cells while MCH cells inhibit orexin neurons; MCH thereby serves as a feedback regulator, exerting an inhibitory influence on orexin signalling to help modulate LH output (Monti et al., 2013).At high concentrations, orexins excite GABAergic interneurons (Liu et al., 2002) and a portion of MCH's sleep inducing properties arise from deactivation of GABAergic neurons (Monti et al., 2013).Given the experimental addition of orexin-A and the absence of a compensatory MCH response, one would expect significant activation of GABAergic circuitrylikely resulting in a reduction in sleep inducing pressure.Moreover, as orexin neurons are under intensive glutamatergic innervation (Henny and Jones, 2006), these changes would lead to significant disruption to GABA/glutamate homeostasis and could therefore exacerbate RmTBI-induced cognitive deficits.
Orexin neurons represent a significant proportion of the projections to the cholinergic neurons within the BF (Villano et al., 2017;Monti et al., 2013).The cholinergic projections from the BF facilitate bottomup sensory processing within the cortex by modulating the response of glutamatergic pyramidal neuronsthis system has been implicated in processes such as synaptic plasticity, learning, memory, and arousalall of which have been related to cortical activation (Villano et al., 2017).We identified a loss of ChAT positive cells in the BF following RmTBI which was unaffected by orexin-A administration.It is therefore possible that the attentional deficits that often arise in response to RmTBI may result from dysfunction between orexin neurons and acetylcholine neurons in the BF.Future studies that employ more nuanced and complex attention-based behavioural measures could answer these questions.
In addition, many of the altered metabolites and metabolomic pathways identified in orexin-A treated RmTBI animals were directly or indirectly linked to the sleep-wake cycle.For example, tryptophan is a precursor to both serotonin and melatonin (Huang et al., 2023); in addition to its antioxidant properties, melatonin is important for synchronizing sleep-wake cycles, with increases in melatonin signalling being associated with the night period (Huang et al., 2023).This is further corroborated by identified changes in melatonin glucuronide, which is a major metabolite of melatonin (Ma et al., 2008).Although serotonin's role on sleep modulation is still controversial, decreased serotonin concentrations due to reduced uptake of tryptophan is speculated to explain some impairments in initiating and maintaining sleep (Humer et al., 2020).Finally, many of the metabolomic pathways that exhibited dysfunction in the orexin and vehicle RmTBI groups were largely related to energetic systems within the brain, specifically those involved in typical and atypical brain fuel sources.Additional glucose and other fuel sources are required following neurological insult to compensate for extraneous demands induced by repair-specific neuronal and glial signalling transmission, actin cytoskeleton remodelling, mitochondrial trafficking, etc., (Zhang et al., 2021).In addition, fatty acids are necessary for cell metabolism, essential components of cell membranes including the BBB, and stimulate angiogenesis post-stroke (Janssen et al., 2021).Taken together, the metabolome results from the CSF samples suggest that animals in the vehicle and orexin-A RmTBI groups were actively undergoing substantial neurological repair at the time of euthanasia.Importantly, within this study, chronic orexin-A administration induced significant changes to components of the sleep-wake cycle following RmTBI, that were not affected in response to chronic modafinil exposure.

Conclusions and future directions
In summary, this study has demonstrated that although both modafinil and orexin-A have wake-promoting properties, when administered chronically, they differentially modify RmTBI outcomes and pathophysiology.While we had hypothesized that both drugs would disrupt glymphatic clearance, and in turn exacerbate negative outcomes from the cumulative insults, this was not the case.Our findings suggest that modafinil's neuroprotective properties are likely independent of its effects on the orexinergic system, since administration of orexin-A produced significantly different outcomes at the behavioural, immunohistochemical, inflammatory, and metabolomic levels.Modafinil appeared to improve behavioural and metabolomic outcomes, but not cellular protein levels as detected by immunohistochemical staining.These findings suggest that modafinil, or at least some of the mechanisms it acts through, have a role in mitigating some of the detrimental consequences of RmTBI.Conversely, orexin-A exhibited some beneficial effects on circulating cytokine levels, suggesting potential antiinflammatory effects, which is consistent with findings from previous research (Couvineau et al., 2019).However, orexin-A administration was detrimental to behaviour, immunohistochemical, and metabolomic outcomes.Overall, these findings underscore the complexity of sleepwake changes in the injured brain while also showcasing the potential for the modification of arousal and sleep systems in the treatment of RmTBI.
As this study exclusively used male rats, it is important for future studies to also examine the effects of RmTBI, modafinil, and orexin-A administration in females.This is particularly important given that orexin has been shown to influence gonadotropin-releasing hormone (GnRH) and luteinizing hormone secretion, with the majority of GnRH neurons expressing OXR1 (Gaskins and Moenter, 2012).Additionally, there are progesterone and estrogen receptors in many sleep-and arousal-promoting areas, such as the LH and LC (Dorsey et al., 2020).Orexin expression is also sexually differentiated with female rats expressing higher concentrations of orexin-A than male rats.Furthermore, while sex steroids in general show enhancement of orexinergic neuron activity, the effects of ovarian sex hormones are more pronounced (Dorsey et al., 2020).Estrogen and progesterone overall appear to promote wakefulness and inhibit sleep in females due to their ability to enhance norepinephrine, serotonin, acetylcholine, dopamine, and histamine activity (Dorsey et al., 2020).Given that most of these neurotransmitters have been implicated in modafinil's 'mechanism' of action (Minzenberg and Carter, 2008), modafinil administration in females would likely result in different outcomes.In addition, future studies could investigate the role of orexins in RmTBI pathophysiology by examining the effects of orexin receptor antagonism on RmTBI outcomes using suvorexant or lemborexant, dual orexin receptor antagonists (Dubey et al., 2015).Since sleep is an important driver of efficient glymphatic clearance, administering a dual orexin receptor antagonist following RmTBI may offer benefit, leading to improvements in behavioural outcomes.Furthermore, considering that we observed more beneficial outcomes associated with modafinil administration, future investigations should delve deeper into identifying which neurotransmitters and neurochemical pathways are responsible for these improvements in RmTBI outcomes.Elucidation of how these arousal-and sleep-promoting systems contribute to RmTBI outcomes will give us better insight into the mechanisms underlying RmTBI pathophysiology, and how to optimize treatment strategies.

Fig. 1 .
Fig. 1.An illustrative experimental timeline depicting the postnatal (P) days that each experimental manipulation and behavioural test battery occurred on.Intracerebroventricular = ICV, mild traumatic brain injury = mTBI.

Fig. 2 .
Fig. 2. Bar graphs displaying behavioural results (acute and chronic), endpoint spleen weight, and brain weight for vehicle and modafinil treated groups.A) Average time-to-right (TTR) following mTBI or sham injury; B) Average time achieved on rotarod (acute); C) Time spent in the open arms of EPM (acute); D) Average time achieved on rotarod (chronic); E) Time spent in the open arms of EPM (chronic); F) Time to reaction on hot plate; G) Time to reaction on cold plate; H) Spleen weight at endpoint; I) Brain weight at endpoint.Mean and SEM are shown.* p < .05.

Fig. 4 .
Fig. 4. Representative images and bar graphs illustrating the quantity of orexin-A positive cell bodies and MCH positive cell bodies in the LH for vehicle and modafinil treated groups.A) Atlas image from Paxinos and Watson (Paxinos and Watson, 2013) with the regions imaged for the LH outlined in red; B) Example of a magnified image of orexin-A labelled cell bodies (magenta) in the right LH -Scale bar represents 100 μm; C) Example of a magnified image of MCH labelled cell bodies (magenta) in the right LH -Scale bar represents 100 μm; D) Average number of orexin-A labelled cell bodies in LH; E) Average number of MCH labelled cell bodies in the LH.Mean and SEM are shown.* p < .05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5 .
Fig. 5. Representative images and bar graphs depicting immunohistochemical results for the BF and LC in vehicle and modafinil treated groups.A) Atlas image from Paxinos and Watson (Paxinos and Watson, 2013) (in red) where the BF images were acquired; B) Example of a magnified image of ChAT labelled cell bodies (magenta) and orexin-A labelled fibres (green) in the right BF -Scale bar represents 100 μm; C) Average number of ChAT labelled cell bodies in the BF; D) Average number of ChAT labelled cell bodies co-localized with orexin-A fibres in the BF; E) Atlas image outlining (in red) where the LC images were acquired; F) Example of a magnified image of TH labelled cell bodies (magenta) and orexin-A labelled fibres (green) in the right LC -Scale bar represents 100 μm; G) Average number of TH labelled cell bodies in the LC; H) Average number of TH labelled cell bodies co-localized with orexin-A fibres in the LC.Mean and SEM are shown.* p < .05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6 .
Fig. 6.Metabolomic findings for vehicle and modafinil treated RmTBI animals.(A) Principal component analysis for vehicle and modafinil treated groups, indicating metabolomic profile variance between samples.(B) Box plot comparing the significantly varied metabolite (myristoleic acid) in vehicle sham animals to modafinil treated RmTBI animals.(C-D) The box plots comparing the significantly varied metabolites in vehicle sham animals to vehicle treated RmTBI animals The y axis for all box plots represents normalised peak intensities obtained from mass spectrometry.

Fig. 7 .
Fig. 7. Bar graphs displaying behavioural results (acute and chronic), endpoint spleen weight, and brain weight for vehicle and orexin-A treated groups.A) Average time-to-right (TTR) following mTBI or sham injury; B) Average time achieved on rotarod (acute); C) Time spent in the open arms of EPM (acute); D) Average time achieved on rotarod (chronic); E) Time spent in the open arms of EPM (chronic); F) Time to reaction on hot plate; G) Time to reaction on cold plate; H) Spleen weight at endpoint; I) Brain weight at endpoint.Mean and SEM are shown.* p < .05.

Fig. 9 .Fig. 10 .
Fig. 9. Representative images and bar graphs illustrating the quantity of orexin-A positive cell bodies and MCH positive cell bodies in the LH for vehicle and orexin-A treated groups.A) Atlas image from Paxinos and Watson (Paxinos and Watson, 2013) with the regions imaged for the LH outlined in red; B) Example of a magnified image of orexin-A labelled cell bodies (magenta) in the left LH -Scale bar represents 100 μm; C) Example of a magnified image of MCH labelled cell bodies (magenta) in the right LH -Scale bar represents 100 μm; D) Average number of orexin-A labelled cell bodies in LH; E) Average number of MCH labelled cell bodies in the LH.Mean and SEM are shown.* p < .05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1
Two-way ANOVA results for circulating cytokine levels for animals treated with modafinil when compared to animals treated with vehicle.Red text denotes significant effects (p < .05).

Table 3
Two-way ANOVA results for circulating cytokine levels when comparing animals treated with orexin-A and animals treated with vehicle.Red text denotes significant effects (p < .05).