Identifying differential responses between sexes following traumatic brain injury (TBI) can elucidate the mechanisms behind disease pathology. Peripheral and central inflammation in the pathophysiology of TBI can increase sleep in male rodents, but this remains untested in females. We hypothesized that diffuse TBI would increase inflammation and sleep in males more so than in females. Diffuse TBI was induced in C57BL/6J mice and serial blood samples were collected (baseline, 1, 5, 7 days post‐injury [DPI]) to quantify peripheral immune cell populations and sleep regulatory cytokines. Brains and spleens were harvested at 7DPI to quantify central and peripheral immune cells, respectively. Mixed‐effects regression models were used for data analysis. Female TBI mice had 77%–124% higher IL‐6 levels than male TBI mice at 1 and 5DPI, whereas IL‐1β and TNF‐α levels were similar between sexes at all timepoints. Despite baseline sex differences in blood‐measured Ly6Chigh monocytes (females had 40% more than males), TBI reduced monocytes by 67% in TBI mice at 1DPI. Male TBI mice had 31%–33% more blood‐measured and 31% more spleen‐measured Ly6G+ neutrophils than female TBI mice at 1 and 5DPI, and 7DPI, respectively. Compared with sham, TBI increased sleep in both sexes during the first light and dark cycles. Male TBI mice slept 11%–17% more than female TBI mice, depending on the cycle. Thus, sex and TBI interactions may alter the peripheral inflammation profile and sleep patterns, which might explain discrepancies in disease progression based on sex.

We used 16-month-old female 3xTg-AD mice and female age-matched C7BL/6;129X1/SvJ;129S1/S wild-type (WT Aged) controls. Mice were ordered from The Jackson Laboratory (Sacramento, CA), were bred at Arizona State University, and were then transferred to the University of Arizona. Mice were acclimated to our laboratory at the University of Arizona for at least two weeks prior to implementing the study. Mice were housed in a 14:10 light-dark cycle (lights on at 6:00AM; 200 lux cool, white fluorescent light) at a constant temperature (23°C ± 2) and given food and water ad libitum, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International.

This study had a 10-day experimental timeline with acclimation starting at day 0 and the final behavioral testing occurring on day 10. Mice were singly housed and acclimated to a non-invasive peizoelectric sleep cage for three days (days 0-2). Following acclimation, baseline sleep was recorded for 48 consecutive hours (days 3-4). On day 5, all mice were removed from their sleep cages and blood was collected, and WT Aged mice received surgical preparation for diffuse brain injury. On day 6, WT Aged mice received a diffuse brain injury via midline fluid percussion injury (mFPI). One-hour post-injury, WT Aged mice were assigned, in order of their sequential identification number, to receive either remote ischemic conditioning (RIC) intervention or an anesthetic control. 3xTg-AD mice were also assigned in order of their sequential identification number to receive either RIC intervention or an anesthetic control on day 6. Thereafter, all mice were immediately returned to their sleep cage and 24 hours of uninterrupted sleep was recorded. On day 7, blood was collected via submandibular vein prior to re-administration of RIC or anesthetic controls in the same groups as described above. Mice were then placed back in sleep cages for 2 days of uninterrupted sleep recording (days 8 and 9). On day 10, mice were tested in an open field and on a novel object recognition task. All experimentation occurred between 8:00AM and 10:00AM to allow for uninterrupted sleep measurements at all other hours, except for day 10 when behavior was evaluated. All behavior testing was conducted in the same room to avoid potential confounding of novel locations on behavioral responses. Sleep data from 16 WT Adult (2-month old) female mice from a separate study (Saber et al., 2019), which were collected under the same conditions as the present study, were used as a control for aging only in comparisons of baseline sleep metrics.

Sleep data were collected using piezoelectric sleep cages (Signal Solutions, Lexington, KY, USA). Monocyte and neutrophil population data were collected from blood samples using flow cytometry. Cytokines data were collected from blood using cytokine assays (MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay).

Correspondence

Rachel K. Rowe, Ph.D.

BARROW Neurological Institute at Phoenix Children’s Hospital

Phoenix Veteran Affairs Health Care System

Translational Neurotrauma Research Program

Department of Child Health

University of Arizona College of Medicine – Phoenix

ABC-1 Building, 425 N. 5^th Street

Phoenix, AZ 85004, USA

Email: rkro222@email.arizona.edu

File List

Sleep.csv

SleepBout_Categories.csv

Blood_Flow.csv

Cytokines.csv

RRT_Weights_Cognitive.csv

File Descriptions

Sleep.csv:  File containing all data from sleep recording via piezoelectric sleep cages.

1. ID – Unique identifier for individual mice that reflects intrinsic nesting (clustering) within four cohort groups (cohort:mouse); for example, mouse 5 in cohort 2 is labeled G2:5.
2. Treatment – Categorical variable denoting whether each mouse received a traumatic brain injury (TBI) via midline fluid percussion or not (None).
3. Genetics – Categorical variable denoting the genetic strain of each mouse; C57BL/6 wild-type (WT) or 3xTg-AD mice to model Alzheimer’s disease (3xtg).
4. Age – Categorical variable denoting the age group of each mouse; 2 month-old (Young) or 16 month-old (Old).
5. Gen_Age – Categorical variable denoting the genetic strain by age of each mouse; 2 month-old wild-type (WTyng), 16 month-old wild-type (WTold), 16 month-old Alzheimer’s disease model (3xtg).
6. Therapy – Categorical variable denoting whether each mouse received remote ischemic conditioning (RIC) intervention or not (No).
7. Cycle – Categorical variable denoting the sleep cycle within which each data point was recorded; first light cycle (LightA), dark cycle (Dark), or second light cycle (LightB).
8. Time – Exact hourly time on 24-hour scale at which each data point was recorded, where 10:00 was the initiation of the 24-hour period and 9:00 was the end of the 24-hour period.
9. Hour – Integer re-specification of the Time variable to inform models of the correct order of each 24-hour period.
10. DPI – Categorical variable denoting days after traumatic brain injury was induced in wild-type mice.
11. BoutR – Bout lengths for each mouse at each hour, measured in integer seconds.
12. PercD – Percentage of time spent sleeping at each hour for each mouse, scaled 0 to 1.
13. Cum_MinsR – Cumulative minutes spent sleeping at each hour for each mouse, measured in integer minutes.

SleepBout_Categories.csv:  File containing all data for sleep bout episode duration categories recorded via piezoelectric sleep cages.

1. ID – Unique identifier for individual mice that reflects intrinsic nesting (clustering) within four cohort groups (cohort:mouse); for example, mouse 5 in cohort 2 is labeled G2:5.
2. Treatment – Categorical variable denoting whether each mouse received a traumatic brain injury (TBI) via midline fluid percussion or not (None).
3. Genetics – Categorical variable denoting the genetic strain of each mouse; C57BL/6 wild-type (WT) or 3xTg-AD mice to model Alzheimer’s disease (3xtg).
4. Age – Categorical variable denoting the age group of each mouse; 2 month-old (Young) or 16 month-old (Old).
5. Gen_Age – Categorical variable denoting the genetic strain by age of each mouse; 16 month-old wild-type (WTold), 16 month-old Alzheimer’s disease model (3xtg).
6. Therapy – Categorical variable denoting whether each mouse received remote ischemic conditioning (RIC) intervention or not (No).
7. DPI – Categorical variable denoting days after traumatic brain injury was induced in wild-type mice.
8. B_Cat – Categorical variable denoting the episode duration category, measured in integer seconds.
9. Num_Bouts_C – Number of bouts in each episode duration category for each mouse.

Blood_Flow.csv:  File containing all data from flow cytometry analysis of monocytes and neutrophils in blood.

1. ID – Unique identifier for individual mice that reflects intrinsic nesting (clustering) within two cohort groups (cohort:mouse); for example, mouse 5 in cohort 2 is labeled G2:5.
2. Treatment – Categorical variable denoting whether each mouse received a traumatic brain injury (TBI) via midline fluid percussion or not (None).
3. Genetics – Categorical variable denoting the genetic strain of each mouse; C57BL/6 wild-type (WT) or 3xTg-AD mice to model Alzheimer’s disease (3xtg).
4. Therapy – Categorical variable denoting whether each mouse received remote ischemic conditioning (RIC) intervention or not (No).
5. DPI – Categorical variable denoting days after traumatic brain injury was induced in wild-type mice.
6. Cd45 – Percentage of CD45⁺ populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
7. Cd11b – Percentage of CD11b⁺ populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
8. Ly6h – Percentage of CD11b⁺Ly6C^high populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
9. Ly6i – Percentage of CD11b⁺Ly6C^int populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
10. Ly6l – Percentage of CD11b⁺Ly6C^low populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
11. Neut – Percentage of CD11b⁺Ly6G⁺ populations for each mouse at each day that blood samples were collected, scaled 0 to 1.
12. Ly6i_Ly6g – Percentage of CD11b⁺Ly6C^intLy6G^– populations for each mouse at each day that blood samples were collected, scaled 0 to 1.

Cytokines.csv:  File containing all data from cytokine assays of blood samples.

1. ID – Unique identifier for individual mice that reflects intrinsic nesting (clustering) within two cohort groups (cohort:mouse); for example, mouse 5 in cohort 2 is labeled G2:5.
2. Treatment – Categorical variable denoting whether each mouse received a traumatic brain injury (TBI) via midline fluid percussion or not (None).
3. Genetics – Categorical variable denoting the genetic strain of each mouse; C57BL/6 wild-type (WT) or 3xTg-AD mice to model Alzheimer’s disease (3xtg).
4. Therapy – Categorical variable denoting whether each mouse received remote ischemic conditioning (RIC) intervention or not (No).
5. DPI – Categorical variable denoting days after traumatic brain injury was induced in wild-type mice.
6. IL6 – Concentration (pg/mL) of IL-6 for each mouse at each day that blood samples were collected.
7. IL1B – Concentration (pg/mL) of IL-1β for each mouse at each day that blood samples were collected.
8. TNFa – Concentration (pg/mL) of TNF-α for each mouse at each day that blood samples were collected.

RRT_Weights_Cognitive.csv: File containing all data from cognitive testing, weight changes, and righting reflexes.

1. ID – Unique identifier for individual mice that reflects intrinsic nesting (clustering) within two cohort groups (cohort:mouse); for example, mouse 5 in cohort 2 is labeled G2:5.
2. Treatment – Categorical variable denoting whether each mouse received a traumatic brain injury (TBI) via midline fluid percussion or not (None).
3. Genetics – Categorical variable denoting the genetic strain of each mouse; C57BL/6 wild-type (WT) or 3xTg-AD mice to model Alzheimer’s disease (3xtg).
4. Therapy – Categorical variable denoting whether each mouse received remote ischemic conditioning (RIC) intervention or not (No).
5. RRT – Righting reflex times for each mouse that received a traumatic brain injury via midline fluid percussion, measured in integer seconds.
6. Time_cent – Time spent in the center of the open field arena for each mouse, measured in integer seconds.
7. Dist_trav – Distance traveled in the open field arena for each mouse, measured in integer centimeters.
8. Discrim_Index – Discrimination index calculated from the time spent exploring old and novel objects in the cognitive testing arena for each mouse, scaled -1 to 1.
9. Base_wt – Baseline weight (g) of each mouse.
10. DPI1_wt – Weight (g) of each mouse that received a traumatic brain injury via midline fluid percussion, one day post-injury.
11. Term_wt – Terminal weight (g) of each mouse at the end of the study (day 10).
12. DDiff_wt – Difference between Base_wt and DPI1_wt for each mouse that received a traumatic brain injury via midline fluid percussion.
13. TDiff_wt – Difference between Base_wt and Term_wt for each mouse.