Lifelong Chronic Sleep Disruption in a Mouse Model of Traumatic Brain Injury

Chronic sleep/wake disturbances (SWDs) are strongly associated with traumatic brain injury (TBI) in patients and are being increasingly recognized. However, the underlying mechanisms are largely understudied and there is an urgent need for animal models of lifelong SWDs. The objective of this study was to develop a chronic TBI rodent model and investigate the lifelong chronic effect of TBI on sleep/wake behavior. We performed repetitive midline fluid percussion injury (rmFPI) in 4-month-old mice and monitored their sleep/wake behavior using the non-invasive PiezoSleep system. Sleep/wake states were recorded before injury (baseline) and then monthly thereafter. We found that TBI mice displayed a significant decrease in sleep duration in both the light and dark phases, beginning at 3 months post-TBI and continuing throughout the study. Consistent with the sleep phenotype, these TBI mice showed circadian locomotor activity phenotypes and exhibited reduced anxiety-like behavior. TBI mice also gained less weight, and had less lean mass and total body water content, compared to sham controls. Further, TBI mice showed extensive brain tissue loss and increased glial fibrillary acidic protein and ionized calcium-binding adaptor molecule 1 levels in the hypothalamus and vicinity of the injury, indicative of chronic neuropathology. In summary, our study identified a critical time window of TBI pathology and associated circadian and sleep/wake phenotypes. Future studies should leverage this mouse model to investigate the molecular mechanisms underlying the chronic sleep/wake phenotypes post-TBI early in life.


Introduction
Sleep/wake disturbances (SWDs) are among the most prevalent long-term consequences in patients suffering from traumatic brain injury (TBI). 1,2The Centers for Disease Control and Prevention estimates that 1.6 to 3.8 million TBIs occur each year in the United States, many of which are left untreated. 3Around 30-70% of patients after a mild TBI experience some form of SWD. 4,5Chronic post-TBI symptoms, such as anxiety, headaches, irritability, and SWD, persist in an estimated 43% of TBI patients that required hospitalization.][18] Lifelong SWD in mouse models has been understudied.The current study aimed to develop the first lifelong chronic TBI model with a central focus on SWD.Through the development of this model, future studies will offer mechanistic insights and treatment options, which ultimately will promote awareness and patient care in the clinic.
In this study, we utilized the murine repetitive (rmFPI) midline fluid percussion injury (mFPI) model to investigate the long-term effects of TBI on the sleep/wake cycle and associated neuropathology. 16,19,20mFPI is one of the most widely used and extensively characterized preclinical TBI models. 21,22Our data brought light to the chronic TBI sequela influencing circadian rhythms and the sleep/wake cycle and may offer new treatment strategies to improve TBI recovery.

Animals
All experiments were conducted in accordance with institutional guidelines and approved by the University of Florida Institutional Animal Care and Use Committee.Four-month-old male C57BL6 mice (The Jackson Laboratory, Bar Harbor, ME) were used for the experiments.All mice were housed under controlled laboratory conditions with a 12-h light/dark schedule (lights on at 7:00 AM and lights off at 7:00 PM), ad libitum access to food and water, temperature 20°C-26°C, and humidity 30-70%.

Repetitive midline fluid percussion injury
All rmFPI surgeries were performed as previously described. 23Mice were anesthetized with 4% isoflurane in 100% oxygen.Each mouse's scalp was shaved and placed in a stereotactic frame fitted with a nose cone to maintain anesthesia with 2% isoflurane in 100% oxygen.A thermostatically controlled heating pad maintained body temperature at 36.5°C-37°C during surgery.A midline sagittal incision exposed the skull from the bregma to the lambda.The skull was cleaned of periosteal connective tissue/fascia and dried using sterile cotton-tipped swabs, and a 3.0-mm craniectomy was made midway between the bregma and the lambda along the sagittal suture without disrupting the underlying dura.The female portion of a Leur-Loc hub removed from a 20-gauge needle was affixed to the craniectomy site using cyanoacrylate.Dental acrylic was then applied around the hub to provide stability.After the dental acrylic hardened, the scalp was sutured around the hub, topical bacitracin ointment was applied to the incision site, and the animal was removed from anesthesia and monitored in a warmed cage until fully ambulatory.
For the induction of injury, each animal was reanesthetized with 4% isoflurane in 100% oxygen for 2 min and the male end of a spacing tube was inserted into the hub.The female end of the hub spacer assembly, filled with normal saline, was attached to the end of the fluid percussion apparatus (Custom Design and Fabrication, Sandston, VA).An injury of mild severity (1.62 -0.08 atm) was administered by releasing a pendulum onto a fluid-filled cylinder to induce a brief fluid pressure pulse upon the intact dura.The pressure pulse measured by the transducer was displayed on a storage oscilloscope (Tektronix TDS2012C), and the peak pressure was recorded.After the injury, animals were visually monitored for recovery of spontaneous respiration and a non-toxic, fast-drying silicone sealant (World Precision Instruments, Sarasota, FL) was applied to the hub to prevent contamination.Additional injuries were repeated at 24 and 48 h after the initial injury respectively.After recovery from the third injury, the hub and dental acrylic were removed and the incision was sutured under anesthesia.Topical bacitracin was then applied to the closed scalp incision.
To alleviate post-surgical discomfort, sustained-release buprenorphine (Wedgewood Pharmacy, Swedesboro, NJ) was subcutaneously injected at the time of surgery and once every 48 h.The duration of transient unconsciousness was determined by measuring the time it took each animal to recover the righting reflex as described before. 24After recovery of the righting reflex, animals were placed in a warmed holding cage to ensure the maintenance of normothermia and monitored during recovery before being returned to the vivarium.For animals receiving a sham injury, all of the above steps were followed with the exception of the release of the pendulum to induce the injury.

Sleep recordings
The non-invasive piezo sleep cage system (Signal Solutions, Lexington, KY) used in this study consisted of 16 individual units that simultaneously monitor sleep and wake states, as previously published. 25This system allows for longitudinal sleep/wake monitoring with greater throughput than electroencephalography/ electromyography.PiezoSleep can assess total, day, and night sleep and wake durations, number of sleep bouts, and sleep bout length.Briefly, sleep was characterized by periodic (3 Hz) and regular amplitude signals recorded from the polyvinylidene fluoride sensors, typical of respiration from a sleeping mouse.In contrast, signals characteristic of wake were both the absence of characteristic sleep signals and higher amplitude, irregular spiking associated with volitional movements.The piezoelectric signals in 2-sec epochs were classified by a linear discriminant classifier algorithm based on frequency and amplitude to assign a binary label of sleep or wake.Mice sleep in a polycyclic manner (>40 sleep episodes per hour) and so mouse sleep was quantified as the minutes spent sleeping per hour, presented as a percentage for each hour.
Data collected from the cage system were clustered into bins over specified periods (e.g., 1 h) using the average of percentage sleep, as well as binned by the length of individual bouts of sleep, and the mean bout lengths were calculated.Sleep data were collected monthly.Hourly percentage sleep was calculated by averaging the percentage of sleep for all days of a given collection by a blinded investigator.

Body composition
Body composition was assessed by nuclear magnetic resonance in conscious mice using the EchoMRI whole body composition analyzer (EchoMedical Systems; EchoMRI, Houston, TX), as previously described. 26

Elevated plus maze test
The elevated plus maze (EPM) apparatus consisted of two darkened and walled (but uncovered) arms and two exposed arms joined by a central square (75.5 • 75.5 • 6 cm), elevated 50.5 cm above the floor.Mice were individually placed in the center square (facing the north closed arm), and activity was tracked for 5 min.A camera placed above the maze recorded the animal's movement and was analyzed using ANY-maze software (version 7.20; Stoelting Co., Wood Dale, IL).

Wheel-running locomotor activity assay
Mice were individually housed in cages equipped with running wheels, and locomotor activity was recorded as we have previously described. 27,28Mice were acclimated to the running wheel cages under a standard 12-h/12-h light/dark cycle for 2 weeks and then released to constant darkness (DD) for 2 weeks.Wheelrunning activity was recorded using the ClockLab 4.011 program (Actimetrics, Wilmette, IL) and analyzed using the ClockLab Analysis 6.0.52 program (Actimetrics).

Histological processing, staining, and lesion volume
Mice were transcardially perfused using a miniperistaltic pump (Fisher Scientific, Pittsburgh, PA) with icecold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) as previously described. 29erfused brains were post-fixed in 4% PFA and then cryoprotected overnight in 15% sucrose followed by 30% sucrose overnight at 4°C.Brains were embedded in a solution of 30% sucrose in Optimal Cutting Temperature medium and stored at À80°C.Tissue sections of 30-lM thickness and 600 lM apart were sectioned using a Leica cryostat (Leica Biosystems, Wetzlar, Germany), starting at À0.5 anterior-posterior (AP) bregma level up to 2.5 AP bregma level.
For the detection of Nissl substance in the cytoplasm of neurons, sections were stained with 0.1% cresyl violet acetate (Electron Microscopy Sciences, Hatfield, PA) for 30 min, following standard methodologies.Scanned coronal sections were analyzed using FIJI v1.54f to measure the percentage of brain tissue loss.Total healthy brain area (HB) and injury area (IA) were calculated to determine the percentage of tissue lost by the impact: Percentage of Tissue Loss = AI HB Healthy brain was characterized by normal architecture and neuronal density after cresyl violet staining.In contrast, injured tissue was determined by loss of neuronal density and tissue reabsorption after chronic TBI.
Borders between healthy and injured tissue were normally sharp and clearly defined.Injury as a percentage of brain area was calculated by adding the uninjured area.Lesion volume (LV) was calculated as the volume (mm 3 ) of a sphere with the estimated injured area: For immunofluorescence detection, sections were washed in PBS then placed in boiling sodium citrate buffer (10 mM of sodium citrate, 0.05% Tween 20; pH 6.0) for 20 min for antigen retrieval.Tissue was washed with PBS and blocked (5% serum, 0.4% Triton X-100 in PBS) for 1 h at room temperature.Sections were then incubated in a humid chamber overnight at 4°C in primary antibody: anti-GFAP (glial fibrillary acidic protein; 1:200 dilution; ab4674; Abcam, Cambridge, MA); anti-Iba1 (ionized calcium-binding adaptor molecule 1; Abcam [ab178846], 1:1000 dilution).Next, tissue was washed in PBS and incubated in fluorochromeconjugated secondary antibody for 1 h at room temperature (Abcam [ab150076, ab150173], 1:500 dilution).Sections were then washed in PBS and mounted and cover-slipped in aqueous mounting media containing 4¢,6-diamidino-2-phenylindole (DAPI).

Western blot
Mice were euthanized by isoflurane overdose followed by cervical dislocation, and brains were dissected in PBS over ice.The sagittal cortex surrounding the injury site and hippocampus were rapidly collected, frozen in liquid nitrogen, and stored at À80°C.Tissues were homogenized with micropestle and motor in 1 • diluted radioimmunoprecipitation assay buffer containing protease inhibitors.Lysate was centrifuged, and the supernatant was collected for protein quantification.Protein extracts were resolved on a 12% sodium dodecyl sulfate/ polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane using the iBlot Ò (Invitrogen, Carlsbad, CA) gel transfer device.Membranes were incubated with antibodies against GFAP (catalog no.: RPCA-GFAP; EnCor Biotechnology, Gainesville, FL) and alpha fodrin/alpha-II spectrin (catalog no.: BML-FG6090-0500; Enzo Life Sciences, Farmingdale, NY).Protein bands were visualized using horseradish peroxidase-conjugated antimouse or antirabbit immunoglobin G (Abcam) and enhanced chemiluminescence (ThermoFisherScientific, Waltham, MA).
Beta-actin detection was used as an internal quantitative control; expression levels of targeted proteins were normalized to those of each investigated protein in the densitometry analysis.Positive signals were detected using the ChemiDoc (Bio-Rad Laboratories, Hercules, CA) imaging system.Densitometric analyses were performed using ImageJ (National Institutes of Health, Bethesda, MD).

Statistical analysis/evaluation
Data are shown as the meanstandard error of the mean and were analyzed using statistical software (GraphPad Prism 9; GraphPad Software Inc., La Jolla, CA).A p value <0.05 was considered statistically significant.A two-tailed t-test was used to compare the sham and TBI groups.

Traumatic brain injury induced a transient loss of righting reflex and reduction in weight gain
After pre-injury baseline sleep recording, 20 agematched mice were randomly assigned to either sham or TBI groups (Fig. 1).Mice were acclimated until 4 months of age, which is considered the mature adult life phase equivalency. 30Surgery was performed in the morning, and mice were allowed to recover for 2-4 h before being subjected to three mFPIs or sham injuries in the afternoon.Each mouse was injured every 24 h apart after the previous TBI or sham injury.There were no differences in fluid percussion injury pressure between the first, second, and third injuries (1.60 -0.04, 1.60 -0.11, and 1.65 -0.08 atm).Subsequent to the final injury, mice were allowed to recover for 2 weeks before beginning monthly sleep recordings in a longitudinal sleep study (Fig. 1).
The righting reflex is the innate tendency of the mouse to return upright after being placed on its side or back.The time duration for loss of righting reflex (LRR) after injury/sham and discontinuation of anesthesia is comparable to the duration of lost consciousness in TBI patients. 313][34][35] On the first day of injury, LRR was considerably longer (366 -53 sec) in rmFPI mice than in shams (138 -110; Fig. 2A).Similarly, on the second day of injury, TBI mice LRR was 166 -93 sec longer than shams (rmFPI 314 -93 sec; sham 148 -92 sec; Fig. 2A).On the third and final day of injury, TBI LRR was 244 -97 sec whereas sham mice LRR was 173 -69 sec (Fig. 2A).][34][35] To evaluate recovery after trauma and overall health condition, we recorded body weight at baseline and daily post-injury until mice recovered back to baseline weight (Fig. 2B).On average, rmFPI mice required more days to recover to baseline body weight compared to sham controls (Fig. 2C).Evaluation of body weight 6 months post-injury revealed that rmFPI mice gained less weight relative to shams despite sharing the same baseline body weight, indicating the long-term effect of TBI on systemic metabolism (Fig. 2D).

Traumatic brain injury mice exhibited sleep deficits beginning at 3 months post-injury
To evaluate the chronic effect of rmTBI on sleep/wake behavior, we used the non-invasive piezoelectric sleep cage system for longitudinal sleep monitoring. 16eginning at 3 months post-injury, a phenotype in sleep duration was observed (Fig. 3A-C).More specifically, significant hourly differences were observed at zeitgeber time (ZT) 10 (during the light phase) as well as ZT13, ZT14, ZT15, and ZT20 (during the dark phase) where rmTBI mice slept less than sham controls (Fig. 3A).Sleep duration was decreased in rmTBI mice during the light phase, dark phase, and total compared to sham controls (Fig. 3B,C).At 3 months post-injury, rmTBI mice spent 430 min sleeping during the light phase compared to 467 min by sham controls (Fig. 3B, left panel).During the dark phase, rmTBI mice slept 201 min compared to 255 min by sham controls (Fig. 3B, right panel).In total, rmTBI mice spent 631 min sleeping compared to 722 min by sham controls; a difference of 91 min per day (Fig. 3C).Before 3 months post-injury, there were no significant differences between groups in the light phase, dark phase, or total sleep amounts (Fig. 3C).Total sleep differences between sham controls and rmTBI mice remained significant on a monthly basis for the remainder of life post-TBI (Fig. 3C).

Traumatic brain injury had a long-term effect on circadian locomotor activity
The sleep/wake cycle is regulated by the central circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus.As a clock output measurement, circadian wheel-running activity generally reflects the function of the SCN clock. 36,37In the presence of a light-dark cycle, mice are generally most active during the dark phase and relatively inactive during the light phase.Endogenous circadian rhythms persist under free-running conditions, such as constant darkness, and can be used to determine circadian behavioral phenotypes.At 17 months post-injury, wheel-running behavior was assessed in mice under constant darkness (Fig. 4A).TBI mice demonstrated a significant increase in wheel-running activity at circadian time (CT) 12 and at CT18-20.This result suggests that TBI had a long-term effect on the SCN clock and influenced circadian locomotor behavior.

Traumatic brain injury mice displayed behavioral disinhibition
Anxiety and depression are psychiatric sequelae often associated with SWD in clinical TBI patients. 38Here, we used the EPM to examine anxiety-like behavior 13 months after the sleep phenotype began.TBI mice entered anxiogenic zones with greater frequency compared to sham controls.The risk-taking behavior is likely attributable to increased impulsivity.At 16 months post-injury, anxiety-like behavior was measured in mice using the EPM.rmTBI mice made a significantly higher proportion of entries into the open arms of the apparatus (41.1% -23.7%) compared to sham controls (15.0%-9.9%; Fig. 4B).The proportion of time spent in anxiogenic zones was marginally increased in TBI mice (32.9% -28.4%) compared to sham controls (12.4% -8.9%).Though rmTBI did not exhibit increased anxiety-like behavior that was observed in other TBI models, they did demonstrate greater risk-taking behavior indicative of a behavioral disinhibition phenotype. 39aumatic brain injury led to changes in body composition later in life post-injury Body-weight discrepancy between rmFPI mice and sham controls persisted at 17 months post-injury (Fig. 4C, left panel).Considering the body mass reduc-tion in rmFPI mice, we measured mice body composition using the EchoMRI system (Fig. 4C).There were no significant differences in fat mass content between the groups (Fig. 4C, left center panel).However, rmFPI mice showed a decrease in lean mass compared to sham controls (Fig. 4C, right center panel).Lean mass is a muscle tissue mass equivalent to all the body parts containing water, excluding fat and bone minerals.This reduction in lean mass may indicate atrophy of muscle or other organs. 40In addition, rmFPI mice exhibited a lower total water content compared to sham controls (Fig. 4C, right panel).These results suggest that the weight reduction in rmFPI mice does not involve a reduction in fat storage, but is likely attributed to muscular and organ atrophy. 41,42

Traumatic brain injury induced chronic lesioning and neuropathology
To evaluate the long-term effect of rmFPI on brain morphology, we performed perfusion and histological analysis of brain sections.Gross inspection of rmFPI brains 19 months post-injury revealed extensive cavitation (Fig. 5A).Affected areas included both hemispheres of the parietal-temporal lobes and part of the frontal lobes, corresponding to the retrosplenial cortex, primary motor cortex, and primary somatosensory cortex.In contrast, sham brains showed no signs of trauma.Cresyl violet coronal sections showed an extension of the injury penetrating up to the thalamus and the anterior areas of both hippocampus (Fig. 5B, left panel).Compared to sham controls, a significant percentage (17.75%) of rmFPI brain tissue was lost because of injury (Fig. 5B, middle panel).Volumetric estimation showed that the injury was 31.48 mm 3 for rmFPI brains and only 0.146 mm 3 for sham (Fig. 5B, right panel).These data showed clear differences of  2).EchoMRI body composition analysis showed no significant changes in fat amount, but a reduction in lean mass and total water in rmFPI mice relative to shams.*p < 0.05; ns, not significant.CT, circadian time; rmFPI, repetitive midline fluid percussion injury; TBI, traumatic brain injury.
histopathology between rmFPI and sham brains.Previous mFPI studies in mice at early time points showed no clear brain tissue loss at gross examination or histological evaluation. 19,43To our knowledge, this is the first study that extends to more than 1 year after injury.
To determine the degree of TBI-induced histopathological alterations in the brain, we used stained tis-sue sections against IBA1 and GFAP to evaluate microglia/macrophage and astrocyte changes. 44,45icroglial activation has been linked to sleep dysregulation in mice and humans, and GFAP plays a critical role in astrogliosis post-injury and neurodegeneration. 46,47We observed an increase in GFAP and IBA1 levels in rmFPI brains, especially in areas adjacent to tissue loss (Fig. 5C).][50][51] Thus, the long-term effect of TBI on the hypothalamus may explain the sleep changes in these rmFPI mice.
Western blot analysis of GFAP level in the cortex and hippocampus further confirmed this finding (Fig. 5E).Further, our western blot data also showed that alpha-II spectrin breakdown product (BDP) was significantly increased in the cortex and slightly increased in the hippocampus of TBI mice (Fig. 5F).Alpha-II spectrin is abundant in neurons, and cleavage into 145/150-kDa BDP by calpain/caspase is indicative of TBI-induced cell death. 52,53Overall, these results show that several brain regions are altered 18 months post-injury, and the pathology data support the sleep phenotypes observed in TBI mice.

Discussion
TBI is a serious public health problem that impacts athletes, civilians, and military personnel worldwide.With reported cases on the rise in recent years, TBI is an important health concern that impacts patients' quality of life over their life span. 54Mild TBIs are the most common, representing *80% of cases. 55SWDs are one of the most prevalent chronic sequelae and, despite occurring in upward of 70% of mild TBI cases, are underrecognized and not well studied. 56lthough many murine studies have investigated sleep post-TBI, the focus has been on the acute phase and not the chronic time points. 16,57To our knowledge, this is the first longitudinal study of chronic SWD in mice.Interestingly, a previous sleep study using mFPI concluded that a diffuse axonal injury model does not lead to chronic SWD in mice. 16However, that study only studied sleep behavior for 5 weeks with a single mFPI and suggested that a secondary injury may be necessary for the induction of chronic sleep disruption in mice.Sleep behavior phenotypes did not develop in our model of rmFPI until 3 months post-injury.This decreased sleep quantity phenotype persisted throughout the life span of the TBI mice.Circadian locomotor activity pattern was consistent with sleep/wake behavior.TBI mice slept less than sham controls and were increasingly active.
It has been hypothesized that sleep is regulated by the central circadian clock located in the SCN and the homeostatic process (whose precise anatomical location is not known). 58,59Whereas the hypothalamus is one of the key sleep homeostatic centers, the SCN is also known to be involved in the regulation of sleep homeostasis.The circadian clock also regulates immune function, and recent studies have established a bidirectional relationship. 49,60,61Further, recent studies from our group have shown that the proinflammatory nuclear factor kappa B (NF-jB) transcription factor directly interacts with the core clock component basic helix-loop-helix ARNT like 1 and interferes with the circadian transcriptional feedback mechanism. 61After TBI, NF-jB is activated within hours in neurons, but it is not detectable after 2 weeks. 62imilarly, constitutively active NF-jB in neurons leads to circadian disruption and sleep dysfunction. 61,63herefore, it is likely that neuronal NF-jB expression could play a role in the acute TBI effects, but its role in the chronic effects is not clear.Meanwhile, NF-jB activation in microglia has been shown to appear within 24 h post-injury, but persists long term. 62,64,65icroglia, as the primary innate immune response, become ''primed'' after a TBI incident and elicit a hyperreactive response to subsequent challenges. 66,67revious studies have suggested that a secondary challenge may be necessary for chronic sleep and behavioral dysfunction. 16,67These long-term neuropathological interactions that disturb sleep through the SCN clock and the hypothalamic homeostatic center are not well understood and warrant future neurobehavioral and mechanistic studies.
Previous studies using repetitive models of TBI have demonstrated a similar trend of shortened LRR times after successive injuries. 68In our study, no significant body weight differences between groups were observed at baseline.However, body weight decreased after each successive injury for both sham and TBI mice, but began to recover 48 h after the third and final hit.Though TBI mice (on average) required an additional day to recover to baseline weight, body weight remained significantly below that of sham controls over their life span.Fat, lean, free water, and total water body composition was measured to further explore these differences.Whereas group housing is the preferred default in the laboratory setting, single housing occurs when necessary to prevent prolonged aggression and severe injury to mice, especially in males. 691][72][73] Therefore, single-housed mice were removed from the body composition assessment.Body composition was measured at the end of life because of chronic differences noted in body weight.Though there were significant differences in fat and free water, TBI mice had less lean mass and total water compared to sham controls.
A limitation of this study was the usage of only male mice and did not explore sex-related differences in females.Future studies will also look at the chronic effects of rmFPI in females.Because of the longitudinal nature of the study, mechanistic approaches could not be utilized, and interventions were kept to a minimum through the end of life.
Further, this study laid a foundation for future investigations into the underlying mechanisms and cause-andeffect relationship between TBI pathology and chronic SWD.Having identified specific time points at which SWDs emerge, further studies can focus on these critical time windows to explore the molecular and cellular changes that contribute to these disruptions.Additionally, exploring new targets and therapeutic approaches to mitigate the SWDs associated with TBI can enhance the overall well-being and quality of life for TBI patients.
Future studies should expand upon these long-term effects, exploring the impact of TBI on sleep in larger cohorts and potentially including diverse populations, including female mice and different TBI severity models.By delving deeper into the complex interactions between TBI pathology, sleep disruption, and associated behavioral and molecular changes, we can gain a more comprehensive understanding of the underlying mechanisms and develop targeted interventions for SWD in TBI patients.
In summary, this study highlights the critical time window of TBI pathogenesis associated with chronic SWD and provides valuable insights for future research.By shedding light on the relationship between TBI and SWD, this work paves the way for further investigations that will ultimately lead to improved long-term solutions for persons living with TBI-related sleep disorders.

Conclusion
In conclusion, this study identified a critical time window of TBI pathology, starting at 3 months postinjury, during which chronic SWD became apparent.By monitoring sleep/wake states in a chronic murine model of TBI, the study revealed a significant decrease in sleep duration during both the light and dark phases, persisting throughout their life span and accompanied by a deterioration in sleep quality.These findings underscore the importance of considering the long-term consequences of TBI on sleep.Future studies will focus on the most appropriate time window of pathophysiology, including the sleep phenotype onset.Future mechanistic studies could offer new targets for intervention, targeting SWD in TBI patients.TBI mice also demonstrated significant decreases in body weight and body composition compared to their sham injury controls.Future studies will explore feeding behaviors and the functional relevance of such differences.

FIG. 1 .
FIG.1.Timeline for chronic sleep evaluation in the rmFPI model.Mice were randomly assigned to either the TBI or sham groups.Baseline sleep was recorded for all mice before surgery.rmFPI or sham injury was performed three times at 24-h intervals after craniectomy and placement of the Luer hub.After recovery, sleep was recorded monthly.rmFPI, repetitive midline fluid percussion injury; TBI, traumatic brain injury.

FIG. 2 .
FIG. 2. rmFPI-induced changes in righting reflex and body weight.(A) rmFPI induced a transient loss of righting reflex after each injury.(B,C) Daily body weight during the first week after injury.TBI mice recovered to baseline body weight more slowly than sham controls.(D) rmFPI mice gained less weight compared to shams 6 months after injury.*p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant.rmFPI, repetitive midline fluid percussion injury; TBI, traumatic brain injury.

FIG. 3 .
FIG. 3. rmFPI mice exhibited sleep disturbances starting at 3 months post-injury.(A) Hourly sleep percentage at 3 months post-injury.Sleep was recorded in a non-invasive piezoelectric sleep system.rmFPI mice showed sleep reduction at ZT10, ZT13, ZT14, ZT15, and ZT20, compared to shams.(B) Sleep percentage and amount at 3 months post-injury.rmFPI mice slept less than sham controls in both the light and dark phases.(C) Monthly sleep duration.Compared to sham controls, rmFPI mice began to show sleep reduction at 3 months and the phenotype continued through 16 months post-TBI.(D) Hourly mean sleep bout lengths at 3 months post-injury.*p < 0.05; **p < 0.01; ns, not significant.rmFPI, repetitive midline fluid percussion injury; TBI, traumatic brain injury; ZT, zeitgeber time.

FIG. 4 .
FIG. 4. rmFPI led to changes in behavior and body composition at 17 months post-injury.(A) Wheelrunning activity in rmFPI mice at 17 months post-injury was significantly increased at CT 12-13 and CT18-21 relative to shams.(B) Behavioral tests showed that rmFPI mice had an increase in the percentage of entries into the open arms at the elevated plus maze test.(C) Body weight and composition.rmFPI mice weighted less than their controls at 17 months post-injury, similar to at 6 months (see Fig.2).EchoMRI body composition analysis showed no significant changes in fat amount, but a reduction in lean mass and total water in rmFPI mice relative to shams.*p < 0.05; ns, not significant.CT, circadian time; rmFPI, repetitive midline fluid percussion injury; TBI, traumatic brain injury.