Diffuse brain injury induces acute post-traumatic sleep

Abstract

Objective: Clinical observations report excessive sleepiness immediately following traumatic brain injury (TBI); however, there is a lack of experimental evidence to support or refute the benefit of sleep following a brain injury. The aim of this study is to investigate acute post-traumatic sleep.

Methods: Sham, mild or moderate diffuse TBI was induced by midline fluid percussion injury (mFPI) in male C57BL/6J mice at 9:00 or 21:00 to evaluate injury-induced sleep behavior at sleep and wake onset, respectively. Sleep profiles were measured post-injury using a non-invasive, piezoelectric cage system. In separate cohorts of mice, inflammatory cytokines in the neocortex were quantified by immunoassay, and microglial activation was visualized by immunohistochemistry.

Results: Immediately after diffuse TBI, quantitative measures of sleep were characterized by a significant increase in sleep (>50%) for the first 6 hours post-injury, resulting from increases in sleep bout length, compared to sham. Acute post-traumatic sleep increased significantly independent of injury severity and time of injury (9:00 vs 21:00). The pro-inflammatory cytokine IL-1β increased in brain-injured mice compared to sham over the first 9 hours post-injury. Iba-1 positive microglia were evident in brain-injured cortex at 6 hours post-injury.

Conclusion: Post-traumatic sleep occurs for up to 6 hours after diffuse brain injury in the mouse regardless of injury severity or time of day. The temporal profile of secondary injury cascades may be driving the significant increase in post-traumatic sleep and contribute to the natural course of recovery through cellular repair.

Figure 1. Schematic of the study design.

Two cohorts of mice were used based on experimental outcome measures: (A) sleep recordings and (B) cortical samples and histology. (A) Mice were acclimated to piezoelectric sleep cages for 8 days while sample sleep recordings were monitored to test signal integrity. All mice received a midline craniotomy one day prior to brain or sham injury. Mice were divided into 2 groups based on the time of day they were subjected to injury (9:00, 21:00). Within each group, mice were selected at random and subjected to sham, mild (0.8 atm) or moderate (1.2–1.3 atm) diffuse brain injury by midline fluid percussion (mFPI) (n = 47). Following injury, mice were placed back into piezoelectric sleep cages and post-traumatic sleep was recorded for 7 days. (B) For biochemistry and histology, mice received a midline craniotomy one day prior to injury or sham injury. Mice were subjected to sham, or moderate (1.2–1.3 atm) diffuse brain injury (9:00) and cortical samples were retrieved at 1, 3, 9, 12, 24, 48, 168 hrs (n = 25). Tissue was also collected and prepared for histology 6 hrs post-injury (n = 3).

Figure 2. Diffuse TBI in the mouse disrupts acute post-traumatic sleep parameters compared to uninjured sham.

(A) A multivariate ANOVA showed a significant increase in mean percent sleep over the first 6 hours post-injury compared to the uninjured sham (mean ±SEM; sham n = 16; injured n = 31; F(1, 45) = 6.545, p = 0.00007). After 6 hours post-injury, the mean percent sleep of injured mice normalized to sham mean percent sleep levels and remained comparable for 7 days post-injury (data not shown). (B) A detailed analysis of the acute post-traumatic sleep (in the first hour) following diffuse TBI indicated a significant time dependent effect on the increase in sleep. A multivariate ANOVA of the rolling average of the mean percent sleep over 5 min intervals showed post-traumatic sleep significantly increased over the first hour post-injury with a significant effect of time (mean ±SEM; sham n = 16; injured n = 31; F(11,495) = 8.22, p<0.0001) and group (mean ±SEM; sham n = 16; injured n = 31; F(1,45) = 37.00, p<0.0001). Bonferroni post hoc analysis was used (*, p<0.05). (C) Acutely post-injury, the brain-injured mice showed an increase in median bout length compared to shams. A multivariate ANOVA revealed an increase in bout length significant over the first 4 hours post-injury (mean ±SEM; sham n = 16; injured n = 31; F(1,45) = 2.9138, p = 0.032). This increase in bout length suggested that the increase in mean percent sleep observed acutely post-injury could result from mice sleeping for longer durations, as opposed to sleeping more bouts after diffuse TBI.

Figure 3. Representative sleep-wake recordings in the first hour post-injury showed sleep bouts interrupted by brief arousal and movement.

Uninjured sham mice showed a periodic rhythm of breathing motion (∼3 Hz) with regular amplitude typical of sleep, interrupted by high frequency and amplitude signals corresponding to movement consistent with an awake mouse (A). Diffuse brain-injured mice showed similar rhythmic breathing classified as sleep interrupted by frequency and amplitude variations corresponding to movement during interbout intervals of sleep (B). The red lines represent the raw piezoelectric sensor data over a one minute (top) or 25 second (bottom) interval. The discontinuous blue line indicates the decision classifier over two second intervals to classify sleep activity from wake activity. The broken green line delineates the threshold (in arbitrary units) to determine sleep activity (above the threshold) from wake activity (below the threshold).

Figure 4. Significant increase in post-traumatic sleep is independent of the time of day of the injury.

Mice subjected to mild or moderate injury at 9:00 (A), following the dark/light transition showed significant increases in acute post-traumatic sleep compared to uninjured sham. A multivariate ANOVA and Bonferroni post-hoc analysis was used (mean ±SEM; sham n = 12; injured n = 17; F(1,25) = 15.95); *, p<0.05). Mice subjected to mild or moderate injury at 21:00 (B), following the light/dark transition also showed significant increases in acute post-traumatic sleep compared to sham. A multivariate ANOVA and Bonferroni post-hoc analysis was used (mean ±SEM; sham n = 5; injured n = 14; F(1,17) = 4.42; *, p<0.05). An increase in sleep is observed acutely following TBI and is observed over the course of the first 3 hours in injured mice compared to sham. After 3 hours, sleep began to normalize in the injured animals and became indistinguishable from sleep in the sham. Mean percent sleep of uninjured sham mice in the 9:00 group was significantly higher than the mean percent sleep of sham mice in the 21:00 group (F(1,15) = 6.303, p = 0.0240), as expected.

Figure 5. The significant increase in post-traumatic sleep is observed acutely following both mild and moderate injury.

A multivariate ANOVA showed a significant increase in mean percent sleep between injured mice and uninjured shams over the first 6-injury with no significant difference between mildly injured mice compared to moderately injured mice (mean ±SEM; sham n = 16; mild n = 16; moderate n = 15; F(2,44) = 3.4773, p = 0.00037).

Figure 6

(A) Temporal profile of IL-1β. The temporal profile indicated that levels in the cortex increase rapidly following moderate injury (9:00) as compared to uninjured sham. Levels of IL-1β peak at or near 9 hours post-injury and return to baseline levels by 12 hours post-injury (One-way ANOVA, mean ±SEM; sham n = 7; injured n = 22; F(7,21) = 6.474; p = 0.0004). Selected comparisons were made (Bonferroni post-hoc), asterisk denotes significance (*, p<0.05) compared to sham. (B, C, D) Microglia morphology, an indicator of microglia activation, was examined after mFPI in the mouse using Iba-1 immunohistochemistry. Iba-1 labels all microglia, however, tissue from a 6 hr sham (40×) (B) compared to a 6 hr mild injury (40×) (C) and a 6 hr moderate injury (40×) (D) show distinct differences in microglia morphology. Microglia in sham (B) demonstrated thin ramified processes (denoted by arrows) strongly contrasting the larger cell bodies and thicker processes (denoted by arrowheads) characteristic of activated microglia observed in the diffuse injured mouse (C, D).