Hypocretin Mediates Sleep and Wake Disturbances in a Mouse Model of Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) is a major cause of disability worldwide. Post-TBI sleep and wake disturbances are extremely common and difficult for patients to manage. Sleep and wake disturbances contribute to poor functional and emotional outcomes from TBI, yet effective therapies remain elusive. A more comprehensive understanding of mechanisms underlying post-TBI sleep and wake disturbance will facilitate development of effective pharmacotherapies. Previous research in human patients and animal models indicates that altered hypocretinergic function may be a major contributor to sleep-wake disturbance after TBI. In this study, we further elucidate the role of hypocretin by determining the impact of TBI on sleep-wake behavior of hypocretin knockout (HCRT KO) mice. Adult male C57BL/6J and HCRT KO mice were implanted with electroencephalography recording electrodes, and pre-injury baseline recordings were obtained. Mice were then subjected to either moderate TBI or sham surgery. Additional recordings were obtained and sleep-wake behavior determined at 3, 7, 15, and 30 days after TBI or sham procedures. At baseline, HCRT KO mice had a significantly different sleep-wake phenotype than control C57BL/6J mice. Post-TBI sleep-wake behavior was altered in a genotype-dependent manner: sleep of HCRT KO mice was not altered by TBI, whereas C57BL/6J mice had more non-rapid eye movement sleep, less wakefulness, and more short wake bouts and fewer long wake bouts. Numbers of hypocretin-positive cells were reduced in C57BL/6J mice by TBI. Collectively, these data indicate that the hypocretinergic system is involved in the alterations in sleep-wake behavior that develop after TBI in this model, and suggest potential therapeutic interventions.

Keywords: TBI; brain injury; controlled cortical impact; orexin; sleep; wake.

FIG. 1.

Schematic representation of the protocols used in the present study. C57BL/6J and hypocretin knockout (HCRT KO) mice were implanted with electroencephalography (EEG) recording electrodes and allowed to recover. Forty-eight hours baseline EEG recordings were obtained from undisturbed mice, after which two mice were dropped from the study due to poor EEG signals. Mice of each genotype were randomized into either control (sham surgeries) or experimental (moderate traumatic brain injury [TBI] surgeries) groups. Moderate TBI was induced using controlled cortical impact with a piston depth of 1.0 mm. Forty-eight–hour recordings were obtained from all mice at 3, 7, 15, and 30 days post-surgery. After the last recording period (32 days post-surgery), animals were perfused and brains were removed for immunohistochemistry.

FIG. 2.

Traumatic brain injury (TBI) decreases wakefulness and increases non-rapid eye movement sleep during the dark period in C57BL/6J mice but not in hypocretin knockout mice. electroencephalography (EEG) and home cage activity recordings were obtained from four groups of mice: sham surgery C57BL/6J mice (n = 8); TBI surgery C57BL/6J mice (n = 9); sham surgery hypocretin knockout (HCRT KO) mice (n = 8); and TBI surgery HCRT KO mice (n = 8). Values are means (± standard error of the mean) percent of time spent in wakefulness (WAKE), non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep during the 12 h dark periods at baseline, 3, 7, 15, and 30 days post-surgery. * indicates a statistically significant difference (p < 0.05) between genotypes (C57BL/6J vs. HCRT KO) within the same condition. # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI) with the same genotype.

FIG. 3.

Traumatic brain injury (TBI) has little impact on wakefulness and sleep during the light period. EEG and home cage activity recordings were obtained from four groups of mice: sham surgery C57BL/6J mice (n = 8); TBI surgery C57BL/6J mice (n = 9); sham surgery hypocretin knockout (HCRT KO) mice (n = 8); and TBI surgery HCRT KO mice (n = 8). Values are means (± SEM) percent of time spent in wakefulness (WAKE), non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep during the 12 h light period at baseline, 3, 7, 15, and 30 days post-surgery. * indicates a statistically significant difference (p < 0.05) between genotypes (C57BL/6J vs. HCRT KO) within the same condition. # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI) with the same genotype.

FIG. 4.

Duration of wake bouts during the dark period is affected by traumatic brain injury (TBI) in a genotype-dependent manner. Wake bouts were sorted into bins based upon their duration. Values are the mean (± SEM) number of bouts in each bin during the 12 h dark periods at baseline, 3, 7, 15, and 30 days post-surgery for C57BL/6J mice with sham surgery (n = 8); C57BL/6J mice subjected to TBI (n = 9); hypocretin knockout (HCRT KO) mice with sham surgery (n = 8); and HCRT KO mice that had TBI (n = 8). * indicates a statistically significant difference (p < 0.05) between genotypes (C57BL/6J vs. HCRT KO) within the same condition. # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI) within the same genotype.

FIG. 5.

Short- and long duration of wake bouts during the light period are affected by traumatic brain injury in a genotype-dependent manner. Wake bouts were sorted into bins based upon their duration. Values are the mean (± SEM) number of bouts in each bin during the 12 h dark periods at baseline, 3, 7, 15, and 30 days post-surgery for C57BL/6J mice with sham surgery (n = 8); C57BL/6J mice subjected to TBI (n = 9); hypocretin knockout (HCRT KO) mice with sham surgery (n = 8); and HCRT KO mice that had TBI (n = 8). * indicates a statistically significant difference (p < 0.05) between genotypes (C57BL/6J vs. HCRT KO) within the same condition. # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI) within the same genotype.

FIG. 6.

Traumatic brain injury (TBI) transiently increases delta power during non-rapid eye movement sleep in C57BL/6J mice. Power in the electroencephalogram (EEG) delta frequency band (0.5–4.5 Hz) was calculated by fast Fourier transform from artifact-free, state-specific epochs during the light and dark periods. Animals with the least artifact from each group were used: C57BL/6J mice with sham surgery (n = 5); C57BL/6J mice subjected to TBI (n = 5); hypocretin knockout (HCRT KO) mice that had sham surgeries (n = 6); and HCRT KO mice with TBI (n = 6). Delta power was normalized as the percent of the total power and is plotted as mean ± standard error of the mean. * indicates a statistically significant difference (p < 0.05) between genotypes (C57BL/6J vs. HCRT KO) within the same condition, whereas # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI) within the same genotype.

FIG. 7.

Traumatic brain injury (TBI) reduces numbers of hypocretin neurons in C57BL/6J mice. Numbers of hypocretin neurons in the lateral hypothalamus ipsilateral to the injury site were estimated using unbiased stereology and the optical fractionator method. Values are means ± standard error of the mean obtained from C57BL/6J mice with sham surgery (n = 8) or TBI (n = 9). Counts were obtained at the end of the study, 32 days post-surgery. # indicates a statistically significant difference (p < 0.05) between conditions (sham vs. TBI). Representative immunofluorescent images of the lateral hypothalamus ipsilateral to the injury site are presented.