Sleep, inflammation, and cognitive behavior of aged wild-type mice subjected to diffuse brain injury and aged 3xTg-AD mice as a model of Alzheimer’s disease

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.

Methods
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).