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. 2024 May 15:38:100797.
doi: 10.1016/j.bbih.2024.100797. eCollection 2024 Jul.

Sleep fragmentation after traumatic brain injury impairs behavior and conveys long-lasting impacts on neuroinflammation

Samuel Houle  1 , Zoe Tapp  1   2 , Shannon Dobres  1 , Sakeef Ahsan  1 , Yvanna Reyes  1 , Christopher Cotter  1 , Jessica Mitsch  1 , Zachary Zimomra  2 , Juan Peng  3 , Rachel K Rowe  4 , Jonathan Lifshitz  5 , John Sheridan  2   6 , Jonathan Godbout  1   2   7 , Olga N Kokiko-Cochran  1   2   7
Affiliations

Sleep fragmentation after traumatic brain injury impairs behavior and conveys long-lasting impacts on neuroinflammation

Samuel Houle et al. Brain Behav Immun Health. .

Abstract

Traumatic brain injury (TBI) causes a prolonged inflammatory response in the central nervous system (CNS) driven by microglia. Microglial reactivity is exacerbated by stress, which often provokes sleep disturbances. We have previously shown that sleep fragmentation (SF) stress after experimental TBI increases microglial reactivity and impairs hippocampal function 30 days post-injury (DPI). The neuroimmune response is highly dynamic the first few weeks after TBI, which is also when injury induced sleep-wake deficits are detected. Therefore, we hypothesized that even a few weeks of TBI SF stress would synergize with injury induced sleep-wake deficits to promote neuroinflammation and impair outcome. Here, we investigated the effects of environmental SF in a lateral fluid percussion model of mouse TBI. Half of the mice were undisturbed, and half were exposed to 5 h of SF around the onset of the light cycle, daily, for 14 days. All mice were then undisturbed 15-30 DPI, providing a period for SF stress recovery (SF-R). Mice exposed to SF stress slept more than those in control housing 7-14 DPI and engaged in more total daily sleep bouts during the dark period. However, SF stress did not exacerbate post-TBI sleep deficits. Testing in the Morris water maze revealed sex dependent differences in spatial reference memory 9-14 DPI with males performing worse than females. Post-TBI SF stress suppressed neurogenesis-related gene expression and increased inflammatory signaling in the cortex at 14 DPI. No differences in sleep behavior were detected between groups during the SF stress recovery period 15-30 DPI. Microscopy revealed cortical and hippocampal IBA1 and CD68 percent-area increased in TBI SF-R mice 30 DPI. Additionally, neuroinflammatory gene expression was increased, and synaptogenesis-related gene expression was suppressed in TBI-SF mice 30 DPI. Finally, IPA canonical pathway analysis showed post-TBI SF impaired and delayed activation of synapse-related pathways between 14 and 30 DPI. These data show that transient SF stress after TBI impairs recovery and conveys long-lasting impacts on neuroimmune function independent of continuous sleep deficits. Together, these finding support that even limited exposure to post-TBI SF stress can have lasting impacts on cognitive recovery and regulation of the immune response to trauma.

Keywords: Fluid percussion injury; Memory; Mouse; Neuroinflammation; Sleep fragmentation.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1
TBI decreases dark period sleep and SF stress fragments dark period sleep 1-7 DPI. (A) Adult C57BL/6 mice received either lateral fluid percussion injury, or sham injury. Following injury, mice were housed in sleep fragmentation (SF) chambers for either 14 or 30 days post injury (DPI). TBI and sham mice either received 5 h of mechanical SF daily from 5am to 10am for the first 14 DPI and then recovered until 30 DPI or did not receive SF as control groups. Sleep was recorded by custom built nonintrusive piezoelectric sensors. Ipsilateral cortices were collected at 14 DPI for RNA analysis via Nanostring nCounter Glial Profiling Panels. Behaviors were assessed 7 DPI (OFT, Y maze), and 14 DPI (MWM) to track functional recovery from TBI. (B) TBI increased righting reflex times (Injury, p < 0.05). Time spent asleep and length of sleep bouts are recorded by piezoelectric sensors. (C) Hourly percent-sleep from the dark period averaged from 1 to 7 DPI showed no differences between groups. (D) Daily percent-sleep from the dark period 1–7 DPI showed no differences between groups within a particular day. (E) Total percent-sleep data was collapsed during the dark period over the first week of SF exposure. TBI decreased the percent-sleep mice engaged in during the dark period 1–7 DPI (Injury, p < 0.05). (F) Sleep bouts from the total dark period of each day 1–7 DPI were measured and sorted into bins depending on length. SF stress increased the number of sleep bouts that mice engaged in between (G) 16–32 s, and (H) 32–64 s. (I) TBI decreased the amount of longer sleep bouts 256–512 s that mice engaged in. (Mean ± SEM) (Percent-sleep = Mean ± SEM, Sleep bouts = Mean ± max/min).
Fig. 2
Fig. 2
TBI prevents SF-dependent increases in sleep bouts 8-14 DPI. We continued to measure sleep from 8 to 14 DPI during the second week that mice were exposed to SF. We found that changes to sleep were isolated to the dark period of each day. (A) We averaged each hour 8–14 DPI to show percent-sleep during each ZT hour of the dark period and found that SF increased percent-sleep (SF, p < 0.05). (B) When looking at daily averages for the entire dark period we found the same main effect where SF increased percent-sleep during the dark period and there is also a day by injury interaction effect where TBI increases percent-sleep specifically 10 DPI (Day x Injury, p < 0.05). (C) Next, we averaged total percent-sleep across 8–14 DPI. Injury and SF interacted to increase percent-sleep in both sham SF and TBI SF mice compared to sham control (Injury x SF, p < 0.05). (D) Next, we analyzed average number of sleep bouts from the total dark period 8–14 DPI. Sham SF mice engaged in more 16–32s (SF, p < 0.05; Injury x SF, p < 0.05; E) and 32–64s (SF, p < 0.05; Injury x SF, p < 0.05; F) sleep bouts than sham control. Sham SF mice engaged in more 64–128s (SF, p < 0.05; Injury x SF, p < 0.05; G) and 128–252s (SF, p < 0.05; Injury x SF, p < 0.05; H) sleep bouts than both sham control and TBI control mice while TBI SF mice were not significantly changed from any group. (I) In longer sleep bouts of 252–512s SF increased the number of sleep bouts mice engaged in during the dark period (SF, p < 0.05). (Percent-sleep = Mean ± SEM, Sleep bouts = Mean ± max/min).
Fig. 3
Fig. 3
Post-Injury SF stress impairs spatial learning and memory 14 DPI. Anxiety-like behavior was tested 7 DPI in the open field test. (A) SF decreased distance traveled in the open field (SF, p < 0.05, but no differences are observed in time spent in the center of the open field (B). Spatial working memory was tested 7 DPI in the Y maze. No differences were observed in total number of arm entries (C), or percent spontaneous alternations (D). Spatial learning and memory were assessed from 9 to 14 DPI using the MWM. (E) TBI impaired performance in the MWM and male TBI SF mice had longer escape latencies than male TBI control mice (Injury, p < 0.05; Injury x SF, p < 0.05). (F) No difference in escape latency were detected in female mice. (G) No differences in swim speed were detected between groups or by sex. (I) Sham control, (J) Sham SF, (K) TBI control, and (L) TBI SF mice swim patterns were tracked and assigned to one of (H) nine spatial search strategies. (M) 14 DPI search strategy usage was quantified and TBI SF mice used more non-spatially directed search strategies than TBI control mice (Injury x SF, p < 0.05. (N) This effect was driven by a higher use of random search strategies 14 DPI by TBI SF mice compared to TBI control mice (Injury x SF, p < 0.05). (Mean ± SEM).
Fig. 4
Fig. 4
SF does not impart lasting deficits in sleep during SF stress recovery period 15–30 DPI. Percent-sleep and sleep bouts were analyzed for the 16 days following the end of daily mechanical SF at 14 DPI. No differences between groups exist 15–21 DPI when comparing hourly (A) or daily (B) percent-sleep. Sleep bouts from 15 to 21 DPI were separated by light (C) and dark period (D) and no differences between groups are displayed. Analysis for (E) average hourly percent-sleep, (F) daily percent-sleep, (G) light period sleep bouts, (H) and dark period sleep bouts, were repeated for 22–29 DPI and no differences are detected. (Percent-sleep = Mean ± SEM, Sleep bouts = Mean ± max/min).
Fig. 5
Fig. 5
Post-TBI SF stress suppresses neurogenesis-related gene expression and increases inflammatory signaling 14 DPI. (A) Heatmap of the standardized z-scores of significantly changed genes constituting the overall effect of TBI in the ipsilateral cortex 14 DPI. Overall effect of TBI was determined by finding genes that were similarly altered in sham vs TBI comparisons within either the control or SF sleep conditions. (B) Heatmap of the standardized z-scores of significantly changed genes that constitute the overall effect of SF in the ipsilateral cortex 14 DPI. Overall effect of SF was determined by finding genes that were similarly altered in control vs SF comparisons within either sham or TBI conditions. (C) Heatmap of the z-scores of genes increased or decreased in TBI SF mice when compared to TBI control mice. (D) Ingenuity Pathway Analysis (IPA) of top upstream regulators that are either inhibited (Activation z-score < −2) or activated (Activation z-score >2) uniquely in TBI SF mice when compared to sham control mice. These upstream regulators were not activated or inhibited by any other condition compared to sham control mice. n = 6/group.
Fig. 6
Fig. 6
Post-TBI SF stress increases IBA1 and CD68 in the ipsilateral cortex and hippocampal CA1 30 DPI. (A) Representative images of IBA1 and CD68 labeling from the lateral cortex ipsilateral to injury. In the merged images inserts are included to show representative IBA1+/CD68+ microglia. (B) IBA1 percent-area was increased by both TBI and SF (Injury, p < 0.05; SF, p < 0.05). TBI SF mice had higher IBA1 precent-area than sham SF mice in the lateral cortex (p < 0.05). (C) SF increased microglial CD68 percent-area withing the lateral cortex (SF, p < 0.05). (D) Representative IBA1 and CD68 images from the hippocampal CA1. (E) TBI and SF (Injury, p < 0.05; SF, p < 0.05) both increased IBA1 percent-area in the CA1. (F) TBI interacted with SF (Injury, p < 0.05; TBI x SF-R, p < 0.05). to increase microglial CD68 percent-area in the CA1 where TBI SF mice had higher microglial CD68 percent-area than all other groups.
Fig. 7
Fig. 7
Post-TBI SF stress conveys lasting changes on inflammatory signaling 30 DPI. (A) Heatmap of the standardized z-scores of differentially expressed genes constituting the overall effect of TBI in the ipsilateral cortex 30 DPI. (B) Heatmap of the standardized z-scores of Prkab2, the only differentially expressed genes constituting the overall effect of SF in the ipsilateral cortex 30 DPI. (C) Heatmap of the z-scores of genes significantly increased or decreased in TBI SF mice when compared to TBI control mice 30 DPI. (D) Ingenuity Pathway Analysis (IPA) of top upstream regulators that are either inhibited (Activation z-score < −2) or activated (Activation z-score >2) uniquely in TBI SF mice when compared to sham control mice. These upstream regulators were not activated or inhibited by any other condition compared to sham control mice. n = 6/group.
Fig. 8
Fig. 8
Post-TBI SF stress impairs and delays the activation of reparative pathways. All pathway enrichment was performed using IPA comparison analysis. (A) IPA comparison analysis showing pathways enriched in TBI control mice and TBI SF mice 14 DPI. (B) IPA comparison analysis showing pathways enriched in TBI control and TBI SF mice 30 DPI. (C) DEGs considered within each pathway to determine pathway enrichment.
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