Microglial depletion and repopulation differentially modulate sleep and inflammation in a mouse model of traumatic brain injury

Abstract

Traumatic brain injury (TBI) causes persistent sleep disturbances, leading to long-term neurological consequences and reduced quality of life. We hypothesized that microglial depletion via PLX5622 (PLX), a colony-stimulating factor 1 receptor (CSFR1R) inhibitor, would exacerbate sleep disturbances and alter inflammatory profiles after TBI, and that microglial repopulation would ameliorate these effects. Male mice received PLX or control diets (21 days) followed by a midline fluid percussion injury (mFPI) or sham surgery. Physiological parameters were recorded non-invasively to determine sleep for 7 days post-injury. Subsequently, PLX was withdrawn to allow microglial repopulation, and sleep was assessed during the 7-day repopulation period. In a subset of mice, repeated blood draws were taken to quantify sleep regulatory cytokine concentrations (interleukin [IL]-6, IL-1β, tumor necrosis factor [TNF]-α). TBI significantly reduced sleep in mice on a control diet during the light period (3, 5, and 7 days post-injury), but not the dark period. In PLX-treated mice, TBI did not alter sleep in the light period, however, sleep in the dark period was increased at 3 days post-injury. During the microglial repopulation period, PLX-treated TBI mice slept significantly more in the dark period compared to PLX sham mice and sleep was similar in control TBI vs PLX TBI mice. Analyses revealed that elimination of microglia did not alter baseline cytokine levels. IL-6 was elevated in PLX TBI mice at 1 and 7 days post-injury compared to TBI mice on control diet, while IL-1β and TNF-α remained unchanged. This study highlights the critical role of microglia in modulating post-TBI sleep and inflammation. Findings suggest differential effects of TBI on sleep depending on microglial depletion or repopulation status, with IL-6 serving as a marker of the inflammatory response in microglia-depleted conditions.

Keywords: Brain injury; Concussion; Cytokines; Inflammation; Interleukin 6; Sleep disturbances.

Fig. 1

Study Design. For the Sleep Study, male mice were acclimated to non-invasive piezoelectric sleep cages for 7 days then randomly assigned to a diet (control, PLX) for 21 days. Following microglial depletion, mice received a midline fluid percussion injury (mFPI), or control sham surgery and physiological parameters were measured for 7 days to determine post-traumatic sleep. At 7 days post-injury, all mice were placed on control diet to allow for microglial repopulation. Sleep was assessed for the duration of the repopulation period. For the Cytokine Study, male mice were acclimated to the vivarium and a baseline blood draw was taken at the initiation of the study. Mice were randomly assigned to a diet (control, PLX) for 21 days. After 21 days of diet administration a blood draw was taken (prior to TBI). Blood was collected at 1 and 7 days post-injury. Pro-inflammatory cytokine levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were assessed.

Fig. 2

TBI altered sleep in mice on control and PLX diets. (A) At 3 days post-injury, TBI significantly decreased sleep during the light period in mice on control diet, and increased sleep in the dark period in the PLX diet group. (B) At 5 days post-injury, disrupted sleep patterns persisted in TBI mice on control diet in the light period. For the PLX diet group, a significant injury × time interaction was observed in the dark period. (C) At 7 days post-injury, TBI-induced sleep disturbances were sustained, with significant effects of injury in both the light and dark periods for mice on control diet, and significant injury × time interaction in the light and dark periods of mice on PLX diet. Hourly percent sleep and total minutes slept are presented as mean ± SEM. Black bars indicate the dark period (zeitgeber time (ZT) 14–23). Significant differences are denoted as follows: ∗ indicates a significant effect of injury, # indicates a significant injury × time interaction, (p < 0.05).

Fig. 3

Sleep was altered in mice with repopulating microglia. PLX diet was withdrawn, and all mice were placed on control diet to allow for repopulation of microglia. (A–B) At 3- and 5 days of microglial repopulation, mice previously on PLX diet that were subjected to TBI slept more than respective shams. (C) At 7 days of repopulation (14-days post-injury), TBI-induced sleep disturbances were noted in the light period of mice on control diet. Hourly percent sleep and total minutes slept are presented as mean ± SEM. Black bars indicate the dark period (zeitgeber time (ZT) 14–23). Significant differences are denoted as follows: ∗ indicates a significant effect of injury, # indicates a significant injury × time interaction, (p < 0.05).

Fig. 4

IL-6 was elevated after TBI in mice on PLX diet. Pro-inflammatory cytokine levels were evaluated at the initiation of the study, following 21 days of administration of control or PLX diet, and at 1- and 7 days post-injury. (A) IL-6 was elevated after TBI in mice on PLX diet compared to mice subjected to TBI on control diet. (B–C) IL-1β and TNF-α levels were similar among groups across all time points. Cytokine levels are presented as mean ± SEM. Significant differences are denoted as follows: ∗ indicates a significant effect, (p < 0.05).