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Abstract

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).^1,2 The 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.³ Around 30–70% of patients after a mild TBI experience some form of SWD.^4,5 Chronic post-TBI symptoms, such as anxiety, headaches, irritability, and SWD, persist in an estimated 43% of TBI patients that required hospitalization. Long-term sleep and circadian disruptions negatively affect life quality and have been associated with diseases, including cancer, diabetes, obesity, hypertension, daytime fatigue, anxiety, and depression, as well as cognitive deficits.^6–9 SWDs are known to contribute to morbidity and long-term health consequences and delay and impair TBI recovery; however, the underlying mechanisms are not well understood.^10–15

Although it is well recognized that TBI early in life is strongly associated with chronic SWD in patients, long-term pre-clinical models and studies are lacking.^16–18 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.^16,19,20 mFPI is one of the most widely used and extensively characterized pre-clinical TBI models.^21,22 Our 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.

Methods

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.²³ Mice 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.²⁴ After 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.²⁵ This 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.^27,28 Mice 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. Wheel-running 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 ice-cold phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) as previously described.²⁹ Perfused 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 −80°C. Tissue sections of 30-μM thickness and 600 μM apart were sectioned using a Leica cryostat (Leica Biosystems, Wetzlar, Germany), starting at −0.5 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: $P e r c e n t a g e o f T i s s u e L o s s = \frac{A I}{H B}$

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 (mm³) of a sphere with the estimated injured area: $L e s i o n V o l u m e = \frac{I A^{\frac{2}{3}}}{6 \sqrt{π}}$

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 fluorochrome-conjugated 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).

For Nissl body staining, GFAP, and Iba1, slides were imaged using a Keyence BZ-X800 microscope (Keyence Corporation, Osaka, Japan) at 4 × magnification. GFAP- and Iba1-stained slides were also imaged using a ZEISS inverted confocal microscope (ZEISS, Oberkochen, Germany) at 10 × or 20 × magnification. Images were processed using ZEN lite 3.8 (ZEISS).

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 −80°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^® (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 mean ± standard 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.

Results

Traumatic brain injury induced a transient loss of righting reflex and reduction in weight gain

After pre-injury baseline sleep recording, 20 age-matched 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.³⁰ Surgery 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).

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.

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.³¹ LRR was used as an index of injury severity in this study as used before.^32–35 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). Overall, sham mice demonstrated a shorter LRR compared to rmFPI mice despite the injury being the same, indicative of the immediate effect of brain trauma and confirming injury in the TBI group as reported in previous studies.^32–35

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.

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.¹⁶ Beginning 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).

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.

At 3 months post-injury, mean sleep bouts were near significance (p = 0.0569) at ZT14, with rmTBI mice averaging 315 ± 116-sec sleep bouts compared to 420 ± 82-sec sleep bouts for sham controls (Fig. 3D). Mean sleep bouts were lower during the light phase (rmFPI 684 ± 129 sec; sham 727 ± 71 sec), dark phase (rmFPI 316 ± 58 sec; sham 334 ± 46 sec), and total (rmFPI 500 ± 75 sec; sham 531 ± 33 sec) for rmTBI compared to sham controls, but not statistically significant. By 16 months post-injury, sleep quality during the light phase worsened (rmFPI 676 ± 207 sec; sham 720 ± 170 sec), but did not reach statistical significance.

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.^36,37 In 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.

FIG. 4.

rmFPI led to changes in behavior and body composition at 17 months post-injury. (A) Wheel-running 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.

Traumatic brain injury mice displayed behavioral disinhibition

Anxiety and depression are psychiatric sequelae often associated with SWD in clinical TBI patients.³⁸ Here, 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.³⁹

Traumatic 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 reduction 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.⁴⁰ In 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 mm³ for rmFPI brains and only 0.146 mm³ for sham (Fig. 5B, right panel). These data showed clear differences of 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.^19,43 To our knowledge, this is the first study that extends to more than 1 year after injury.

FIG. 5.

rmFPI-induced damage of cortex and hippocampus associated with glial activation. (A) Gross image of post-mortem brains showing extensive cavitation. (B) Cresyl violet coronal brain sections showing that the injury extended to the thalamus and hippocampus. Quantification of the lesion (31.48 mm³) represents 17.75% of the brain area of rmFPI mice, compared to 0.146 mm³ for sham controls. (C) Immunofluorescence images of GFAP and IBA1 staining. rmFPI brains showed an increase in GFAP and IBA1 levels, especially in areas adjacent to the injury site. (D) Confocal images revealed an increase in GFAP and IBA1 signals in the cortex, hippocampus, PVN, and SCN of rmFPI brains compared to shams. (E,F) Western blot analysis of GFAP (E) and alpha-II spectrin (F) in the cortex and hippocampus of sham and TBI mice. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant. A-P, anterior-posterior; BDP, breakdown product; BL, bilateral; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; IBA1, ionized calcium-binding adaptor molecule 1; rmFPI, repetitive midline fluid percussion injury; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; TBI, traumatic brain injury.

To determine the degree of TBI-induced histopathological alterations in the brain, we used stained tissue sections against IBA1 and GFAP to evaluate microglia/macrophage and astrocyte changes.^44,45 Microglial activation has been linked to sleep dysregulation in mice and humans, and GFAP plays a critical role in astrogliosis post-injury and neurodegeneration.^46,47 We observed an increase in GFAP and IBA1 levels in rmFPI brains, especially in areas adjacent to tissue loss (Fig. 5C). Higher magnification confocal images revealed an increase in GFAP and IBA1 signals in the cortex, hippocampus, and hypothalamus (more specifically, the paraventricular nucleus [PVN] and SCN, known to regulate circadian rhythms and the sleep/wake cycle) of rmFPI brains (Fig. 5D).^48–51 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.^52,53 Overall, 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.⁵⁴ Mild TBIs are the most common, representing ∼80% of cases.⁵⁵ SWDs are one of the most prevalent chronic sequelae and, despite occurring in upward of 70% of mild TBI cases, are under-recognized and not well studied.⁵⁶

Although many murine studies have investigated sleep post-TBI, the focus has been on the acute phase and not the chronic time points.^16,57 To 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.¹⁶ However, 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).^58,59 Whereas 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.^49,60,61 Further, recent studies from our group have shown that the proinflammatory nuclear factor kappa B (NF-κB) transcription factor directly interacts with the core clock component basic helix-loop-helix ARNT like 1 and interferes with the circadian transcriptional feedback mechanism.⁶¹ After TBI, NF-κB is activated within hours in neurons, but it is not detectable after 2 weeks.⁶²

Similarly, constitutively active NF-κB in neurons leads to circadian disruption and sleep dysfunction.^61,63 Therefore, it is likely that neuronal NF-κB expression could play a role in the acute TBI effects, but its role in the chronic effects is not clear. Meanwhile, NF-κB activation in microglia has been shown to appear within 24 h post-injury, but persists long term.^62,64,65 Microglia, as the primary innate immune response, become “primed” after a TBI incident and elicit a hyper-reactive response to subsequent challenges.^66,67

Previous studies have suggested that a secondary challenge may be necessary for chronic sleep and behavioral dysfunction.^16,67 These 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.⁶⁸ In 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.⁶⁹ However, single housing has been shown to decrease body mass and increase adiposity, compared to group housing.^70–73 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-and-effect 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 post-injury, 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.

Footnotes

Acknowledgments

We acknowledge funding from the National Institutes of Health (1R21AA029785-01A1). We also express our gratitude to the University of Florida Claude D. Pepper Older Americans Independence Center (OAIC) Circadian Rhythms research core for allowing us to use the sleep and activity recording equipment. We also thank Dr. Karyn A. Esser for use of the EchoMRI whole body composition analyzer and Drs. Belinda S. Pinto and Eric T. Wang for use of the ZEISS inverted confocal microscope.

Authors' Contributions

Andrew Morris: conceptualization, methodology, formal analysis, investigation, data curation, writing–original draft, writing–review & editing, visualization, project administration. Erwin K. Gudenschwager Basso: methodology, formal analysis, data curation, writing–review & editing. Miguel A. Gutierrez-Monreal: writing–review & editing. R. Daniel Arja: investigation, writing–review & editing. Firas H. Kobeissy: formal analysis, writing–review & editing, supervision. Christopher G. Janus: formal analysis, resources, writing–review & editing, supervision. Kevin K.W. Wang: resources. Jiepei Zhu: resources, investigation, writing–review & editing. Andrew C. Liu: resources, writing–review & editing, supervision, funding acquisition.

Funding Information

We acknowledge funding from the National Institutes of Health (NIH) National Institute on Alcohol Abuse and Alcoholism (NIAAA; 5R21AA029785-02).

Author Disclosure Statement

No competing financial interests exist.

Abbreviations Used

References

Barshikar

, Bell

. Sleep disturbance after TBI. Curr Neurol Neurosci Rep, 2017; 17(11):87; doi: 10.1007/s11910-017-0792-4

Rowe

, Griesbach

. Immune-endocrine interactions in the pathophysiology of sleep-wake disturbances following traumatic brain injury: a narrative review. Brain Res Bull, 2022; 185:117–128; doi: 10.1016/j.brainresbull.2022.04.017

Langlois

, Rutland-Brown

, Wald

. The epidemiology and impact of traumatic brain injury: a brief overview. J Head Trauma Rehabil, 2006; 21(5):375–378; doi: 10.1097/00001199-200609000-00001

Ouellet

, Savard

, Morin

. Insomnia following traumatic brain injury: a review. Neurorehabil Neural Repair, 2004; 18(4):187–198; doi: 10.1177/1545968304271405

Viola-Saltzman

, Watson

. Traumatic brain injury and sleep disorders. Neurol Clin, 2012; 30(4):1299–1312; doi: 10.1016/j.ncl.2012.08.008

Cantor

, Bushnik

, Cicerone

, et al. Insomnia, fatigue, and sleepiness in the first 2 years after traumatic brain injury: an NIDRR TBI model system module study. J Head Trauma Rehabil, 2012; 27(6):E1–E14; doi: 10.1097/HTR.0b013e318270f91e

Noh

. The effect of circadian and sleep disruptions on obesity risk. J Obes Metab Syndr, 2018; 27(2):78–83; doi: 10.7570/jomes.2018.27.2.78

Shafi

, Knudsen

. Cancer and the circadian clock. Cancer Res, 2019; 79(15):3806–3814; doi: 10.1158/0008-5472.CAN-19-0566

Walker

, Walton

, DeVries

, et al. Circadian rhythm disruption and mental health. Transl Psychiatry, 2020; 10(1):28; doi: 10.1038/s41398-020-0694-0

10.

Kalmbach

, Conroy

, Falk

, et al. Poor sleep is linked to impeded recovery from traumatic brain injury. Sleep, 2018; 41(10):zsy147; doi: 10.1093/sleep/zsy147

11.

Fleming

, Smejka

, Henderson Slater

, et al. Sleep disruption after brain injury is associated with worse motor outcomes and slower functional recovery. Neurorehabil Neural Repair, 2020; 34(7):661–671; doi: 10.1177/1545968320929669

12.

Sweeney

, Peart

, Kyza

, et al. Impaired insulin profiles following a single night of sleep restriction: the impact of acute sprint interval exercise. Int J Sport Nutr Exerc Metab, 2020; 30(2):139–144; doi: 10.1123/ijsnem.2019-0235

13.

Svensson

, Saito

, Svensson

, et al. Association of sleep duration with all- and major-cause mortality among adults in Japan, China, Singapore, and Korea. JAMA Netw Open, 2021; 4(9):e2122837; doi: 10.1001/jamanetworkopen.2021.22837

14.

Gangitano

, Martinez-Sanchez

, Bellini

, et al. Weight loss and sleep, current evidence in animal models and humans. Nutrients, 2023; 15(15):3431; doi: 10.3390/nu15153431

15.

Henríquez-Beltrán

, Dreyse

, Jorquera

, et al. The U-shaped association between sleep duration, all-cause mortality and cardiovascular risk in a Hispanic/Latino clinically based cohort. J Clin Med, 2023; 12(15):4961; doi: 10.3390/jcm12154961

16.

Rowe

, Harrison

, O'Hara

, et al. Diffuse brain injury does not affect chronic sleep patterns in the mouse. Brain Inj, 2014; 28(4):504–510; doi: 10.3109/02699052.2014.888768

17.

Büchele

, Morawska

, Schreglmann

, et al. Novel rat model of weight drop-induced closed diffuse traumatic brain injury compatible with electrophysiological recordings of vigilance states. J Neurotrauma, 2016; 33(13):1171–1180; doi: 10.1089/neu.2015.4001

18.

Tapp

, Kumar

, Witcher

, et al. Sleep disruption exacerbates and prolongs the inflammatory response to traumatic brain injury. J Neurotrauma, 2020; 37(16):1829–1843; doi: 10.1089/neu.2020.7010

19.

Witcher

, Dziabis

, Bray

, et al. Comparison between midline and lateral fluid percussion injury in mice reveals prolonged but divergent cortical neuroinflammation. Brain Res, 2020; 1746:146987; doi: 10.1016/j.brainres.2020.146987

20.

Bray

, Witcher

, Adekunle-Adegbite

, et al. Chronic cortical inflammation, cognitive impairment, and immune reactivity associated with diffuse brain injury are ameliorated by forced turnover of microglia. J Neurosci, 2022; 42(20):4215–4228; doi: 10.1523/JNEUROSCI.1910-21.2022

21.

Xiong

, Mahmood

, Chopp

. Animal models of traumatic brain injury. Nat Rev Neurosci, 2013; 14(2):128–142; doi: 10.1038/nrn3407

22.

Lyeth

. Historical review of the fluid-percussion TBI model. Front Neurol, 2016; 7:217; doi: 10.3389/fneur.2016.00217

23.

Ogino

, Vascak

, Povlishock

. Intensity specific repetitive mild traumatic brain injury evokes an exacerbated burden of neocortical axonal injury. J Neuropathol Exp Neurol, 2018; 77(9):782–792; doi: 10.1093/jnen/nly054

24.

Ogino

, Bernas

, Greer

, et al. Axonal injury following mild traumatic brain injury is exacerbated by repetitive insult and is linked to the delayed attenuation of NeuN expression without concomitant neuronal death in the mouse. Brain Pathol, 2022; 32(2):e13034; doi: 10.1111/bpa.13034

25.

Donohue

, Medonza

, Crane

, et al. Assessment of a non-invasive high-throughput classifier for behaviours associated with sleep and wake in mice. Biomed Eng Online, 2008; 7:14; doi: 10.1186/1475-925X-7-14

26.

Harfmann

, Schroder

, Kachman

, et al. Muscle-specific loss of Bmal1 leads to disrupted tissue glucose metabolism and systemic glucose homeostasis. Skelet Muscle, 2016; 6:12; doi: 10.1186/s13395-016-0082-x

27.

Ramanathan

, Kathale

, Liu

, et al. mTOR signaling regulates central and peripheral circadian clock function. PLoS Genet, 2018; 14(5):e1007369; doi: 10.1371/journal.pgen.1007369

28.

Shen

, Endale

, Wang

, et al. NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1. PLoS Genet, 2021; 17(11):e1009933; doi: 10.1371/journal.pgen.1009933

29.

Stickrod

, Stansifer

. Perfusion of small animals using a mini-peristaltic pump. Physiol Behav, 1981; 27(6):1127–1128; doi: 10.1016/0031-9384(81)90382-6

30.

Fox

, Barthold

, Davisson

, et al., (eds). The Mouse in Biomedical Research, 2nd Edition. Academic: London; 2007; doi: 10.1142/9781860947162_0015

31.

Berman

, Spencer

, Boese

, et al. Loss of consciousness and righting reflex following traumatic brain injury: predictors of post-injury symptom development (a narrative review). Brain Sci, 2023; 13(5):750; doi: 10.3390/brainsci13050750

32.

Greer

, McGinn

, Povlishock

. Diffuse traumatic axonal injury in the mouse induces atrophy, c-Jun activation, and axonal outgrowth in the axotomized neuronal population. J Neurosci, 2011; 31(13):5089–5105; doi: 10.1523/JNEUROSCI.5103-10.2011

33.

Greer

, Hånell

, McGinn

, et al. Mild traumatic brain injury in the mouse induces axotomy primarily within the axon initial segment. Acta Neuropathol, 2013; 126(1):59–74; doi: 10.1007/s00401-013-1119-4

34.

Hånell

, Greer

, McGinn

, et al. Traumatic brain injury-induced axonal phenotypes react differently to treatment. Acta Neuropathol, 2015; 129(2):317–332; doi: 10.1007/s00401-014-1376-x

35.

Vascak

, Jin

, Jacobs

, et al. Mild traumatic brain injury induces structural and functional disconnection of local neocortical inhibitory networks via parvalbumin interneuron diffuse axonal injury. Cerebral Cortex, 2018; 28(5):1625–1644; doi: 10.1093/cercor/bhx058

36.

Verwey

, Robinson

, Amir

. Recording and analysis of circadian rhythms in running-wheel activity in rodents. J Vis Exp, 2013;(71):50186; doi: 10.3791/50186

37.

Healy

, Morris

, Liu

. Circadian synchrony: sleep, nutrition, and physical activity. Front Netw Physiol, 2021; 1: :732243; doi: 10.3389/fnetp.2021.732243

38.

Baumann

. Traumatic brain injury and disturbed sleep and wakefulness. Neuromolecular Med, 2012; 14(3):205–212; doi: 10.1007/s12017-012-8178-x

39.

Tucker

, McCabe

. Measuring anxiety-like behaviors in rodent models of traumatic brain injury. Front Behav Neurosci, 2021; 15:682935; doi: 10.3389/fnbeh.2021.682935

40.

Shahidi

, Shah

, Esparza

, et al. Skeletal muscle atrophy and degeneration in a mouse model of traumatic brain injury. J Neurotrauma, 2018; 35(2):398–401; doi: 10.1089/neu.2017.5172

41.

JafariNasabian

, Inglis

, Reilly

, et al. Aging human body: changes in bone, muscle and body fat with consequent changes in nutrient intake. J Endocrinol, 2017; 234(1):R37–R51; doi: 10.1530/JOE-16-0603

42.

Reynolds

, Dalton

, Calzini

, et al. The impact of age and sex on body composition and glucose sensitivity in C57BL/6J mice. Physiol Rep, 2019; 7(3):e13995; doi: 10.14814/phy2.13995

43.

Ogino

, Vascak

, Povlishock

. Intensity specific repetitive mild traumatic brain injury evokes an exacerbated burden of neocortical axonal injury. J Neuropathol Exp Neurol, 2018; 77(9):782–792; doi: 10.1093/jnen/nly054

44.

Imai

, Ibata

, Ito

, et al. A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun, 1996; 224(3):855–862; doi: 10.1006/bbrc.1996.1112

45.

Yang

, Wang

. Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci, 2015; 38(6):364–374; doi: 10.1016/j.tins.2015.04.003

46.

Bellesi

, de Vivo

, Chini

, et al. Sleep loss promotes astrocytic phagocytosis and microglial activation in mouse cerebral cortex. J Neurosci, 2017; 37(21):5263–5273; doi: 10.1523/JNEUROSCI.3981-16.2017

47.

Das

, Tang

, Han

, et al. CCL20-CCR6 axis modulated traumatic brain injury-induced visual pathologies. J Neuroinflammation, 2019; 16(1):115; doi: 10.1186/s12974-019-1499-z

48.

Yoo

, Yamazaki

, Lowrey

, et al. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci U S A, 2004; 101(15):5339–5346; doi: 10.1073/pnas.0308709101

49.

Morris

, Stanton

, Roman

, et al. Systems level understanding of circadian integration with cell physiology. J Mol Biol, 2020; 432(12):3547–3564; doi: 10.1016/j.jmb.2020.02.002

50.

Yamakawa

, Brady

, Sun

, et al. The interaction of the circadian and immune system: desynchrony as a pathological outcome to traumatic brain injury. Neurobiol Sleep Circadian Rhythms, 2020; 9:100058; doi: 10.1016/j.nbscr.2020.100058

51.

Jiang

, Chen

, Huang

, et al. Role of the paraventricular nucleus of the hypothalamus in sleep–wake regulation. Brain Sci Adv, 2022; 8(3):197–209; doi: 10.26599/BSA.2022.9050017

52.

Weiss

, Wang

KKW

, Allen

, et al. Alpha II-spectrin breakdown products serve as novel markers of brain injury severity in a canine model of hypothermic circulatory arrest. Ann Thorac Surg, 2009; 88(2):543–550; doi: 10.1016/j.athoracsur.2009.04.016

53.

Berger

, Hayes

, Richichi

, et al. Serum concentrations of ubiquitin C-terminal hydrolase-L1 and αII-spectrin breakdown product 145 kDa correlate with outcome after pediatric TBI. J Neurotrauma, 2012; 29(1):162–167; doi: 10.1089/neu.2011.1989

54.

Taylor

, Bell

, Breiding

, et al. Traumatic brain injury-related emergency department visits, hospitalizations, and deaths—United States, 2007 and 2013. MMWR Surveill Summ, 2017; 66(9):1–16; doi: 10.15585/mmwr.ss6609a1

55.

Katz

, Cohen

, Alexander

. Mild traumatic brain injury. Handb Clin Neurol, 2015; 127:131–156; doi: 10.1016/B978-0-444-52892-6.00009-X

56.

Viola-Saltzman

, Watson

. Traumatic brain injury and sleep disorders. Neurol Clin, 2012; 30(4):1299–1312; doi: 10.1016/j.ncl.2012.08.008

57.

Rowe

, Striz

, Bachstetter

, et al. Diffuse brain injury induces acute post-traumatic sleep. PLoS One, 2014; 9(1):e82507; doi: 10.1371/journal.pone.0082507

58.

Saper

, Scammell

, Lu

. Hypothalamic regulation of sleep and circadian rhythms. Nature, 2005; 437(7063):1257–1263; doi: 10.1038/nature04284

59.

Deboer

. Sleep homeostasis and the circadian clock: do the circadian pacemaker and the sleep homeostat influence each other's functioning?. Neurobiol Sleep Circadian Rhythms, 2018; 5:68–77; doi: 10.1016/j.nbscr.2018.02.003

60.

Bellet

, Zocchi

, Sassone-Corsi

. The RelB subunit of NFκB acts as a negative regulator of circadian gene expression. Cell Cycle, 2012; 11(17):3304–3311; doi: 10.4161/cc.21669

61.

Shen

, Endale

, Wang

, et al. NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1. PLoS Genet, 2021; 17(11):e1009933; doi: 10.1371/journal.pgen.1009933

62.

Nonaka

, Chen

, Pierce

, et al. Prolonged activation of NF-kappaB following traumatic brain injury in rats. J Neurotrauma, 1999; 16(11):1023–1034; doi: 10.1089/neu.1999.16.1023

63.

Lee

, Endale

, Wu

, et al. Integration of genome-scale data identifies candidate sleep regulators. Sleep, 2023; 46(2):zsac279; doi: 10.1093/sleep/zsac279

64.

Witcher

, Eiferman

, Godbout

. Priming the inflammatory pump of the CNS after traumatic brain injury. Trends Neurosci, 2015; 38(10):609–620; doi: 10.1016/j.tins.2015.08.002

65.

Donat

, Scott

, Gentleman

, et al. Microglial activation in traumatic brain injury. Front Aging Neurosci, 2017; 9:208; doi: 10.3389/fnagi.2017.00208

66.

Puntambekar

, Saber

, Lamb

, et al. Cellular players that shape evolving pathology and neurodegeneration following traumatic brain injury. Brain Behav Immun, 2018; 71:9–17; doi: 10.1016/j.bbi.2018.03.033

67.

Kokiko-Cochran

, Godbout

. The inflammatory continuum of traumatic brain injury and Alzheimer's disease. Front Immunol, 2018; 9:672; doi: 10.3389/fimmu.2018.00672

68.

Kahriman

, Bouley

, Smith

, et al. Mouse closed head traumatic brain injury replicates the histological tau pathology pattern of human disease: characterization of a novel model and systematic review of the literature. Acta Neuropathol Commun, 2021; 9(1):118; doi: 10.1186/s40478-021-01220-8

69.

National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. National Academies Press: Washington, DC; 2011; doi: 10.17226/12910

70.

Nagy

, Krzywanski

, Li

, et al. Effect of group vs. single housing on phenotypic variance in C57BL/6J mice. Obes Res, 2002; 10(5):412–415; doi: 10.1038/oby.2002.57

71.

Pasquarelli

, Voehringer

, Henke

, et al. Effect of a change in housing conditions on body weight, behavior and brain neurotransmitters in male C57BL/6J mice. Behav Brain Res, 2017; 333:35–42; doi: 10.1016/j.bbr.2017.06.018

72.

Schipper

, Harvey

, van der Beek

, et al. Home alone: a systematic review and meta-analysis on the effects of individual housing on body weight, food intake and visceral fat mass in rodents. Obes Rev, 2018; 19(5):614–637; doi: 10.1111/obr.12663

73.

Sun

, Choi

, Magee

, et al. Metabolic effects of social isolation in adult C57BL/6 mice. Int Sch Res Notices, 2014; 2014:690950; doi: 10.1155/2014/690950

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

Abstract

Introduction

Methods

Animals

Repetitive midline fluid percussion injury

Sleep recordings

Body composition

Elevated plus maze test

Wheel-running locomotor activity assay

Histological processing, staining, and lesion volume

Western blot

Statistical analysis/evaluation

Results

Traumatic brain injury induced a transient loss of righting reflex and reduction in weight gain

Traumatic brain injury mice exhibited sleep deficits beginning at 3 months post-injury

Traumatic brain injury had a long-term effect on circadian locomotor activity

Traumatic brain injury mice displayed behavioral disinhibition

Traumatic brain injury led to changes in body composition later in life post-injury

Traumatic brain injury induced chronic lesioning and neuropathology

Discussion

Conclusion

Footnotes

Acknowledgments

Authors' Contributions

Funding Information

Author Disclosure Statement

Abbreviations Used

References