DOI: 10.5061/dryad.tdz08kq3p
title: Gene expression and ELISA data

description: Obstructive sleep apnea is increasing worldwide, leading to disordered
 sleep patterns and inflammatory responses in brain and peripheral tissues
 that predispose individuals to chronic disease. Pro-inflammatory cytokines
 activate the inflammatory response and their increased expression is
 observed among mice exposed to experimental sleep fragmentation.
 Additionally, glucocorticoids are often secreted from the adrenal cortices
 during sleep loss and are well known to regulate inflammation. However,
 the temporal dynamics of inflammatory responses and
 hypothalamic-pituitary-adrenal (HPA) axis activation in relation to acute
 sleep fragmentation (ASF; 1-24h) are unknown. Male C57BL/6J mice were
 exposed to ASF or control conditions (no ASF) over specified time
 intervals (1, 2, 6, and 24 h) to elucidate the timing of onset of
 glucocorticoid release and pro-inflammatory responses. Cytokine gene
 expression (IL-1b, TNF-a) in brain and peripheral tissues and serum
 glucocorticoid and interleukin-6 (IL-6) concentration were measured to
 determine the temporal relationships of neuroendocrine-immune responses to
 acute SF. The HPA axis was rapidly activated, leading to elevated serum
 corticosterone from 1-24 h of ASF compared with controls. This activation
 was followed by elevated serum IL-6 concentration from 2–24 h of ASF. The
 tissue to first exhibit increased pro-inflammatory gene expression from
 ASF was the heart (1 h of ASF). In contrast, pro-inflammatory gene
 expression was suppressed in the hypothalamus after 1 h of ASF, but
 elevated after 6 h. Because the HPA axis was elevated throughout ASF, this
 suggests that brain, but not peripheral, pro-inflammatory responses were
 rapidly inhibited by glucocorticoid immunosuppression. Instead, activation
 of the sympathetic nervous system (SNS) may potentiate inflammation in the
 short-term for peripheral tissues, but further empirical studies are
 needed. Understanding the mechanisms underlying the onset of inflammatory
 responses to sleep fragmentation could provide new therapeutic options to
 individuals coping with sleep disorders.
description: <strong>Animals.
 </strong>Male adult C57BL/6J mice between 8-12 weeks
 of age were used in this study (<em>n</em>=110; Jackson Laboratory, Bar Harbor,
 ME).  Mice were given food and water <em>ad
 libitum</em> and housed under standard rodent colony
 conditions (lights on: 0800-2000 h, 21<sup>°</sup>C ±
 1<sup>°</sup>C) at Western Kentucky University. Acute
 sleep fragmentation (ASF) experiments were
 performed using automated sleep fragmentation chambers (Lafayette
 Instrument Company; Lafayette, IN; model 80390) with a thin layer of corn
 bedding as previously described and each chamber contained no more than
 five mice. These chambers ensure that mice are
 subjected to sleep fragmentation and not absolute sleep
 deprivation. Mice were acclimated to the sleep
 fragmentation (SF) chambers for 48 h before the commencement of
 experiments to minimize carryover effects from the different cage
 environments.  This study was conducted under the
 approval of the Institutional Animal Care and Use Committee at Western
 Kentucky University (#19-11), and procedures followed the National
 Institutes of Health’s “Guide for the Use and Care of Laboratory Animals”
 and ARRIVE guidelines. <strong> </strong> <strong>Acute sleep fragmentation (Acute SF) and sample collection.      </strong>Starting at 0800 (lights on), mice were exposed to 1, 2, 6, 12, or 24 h (n=110; all groups,<em> n</em> = 10) of ASF, which involves a sweeping bar that moves horizontally across the modified cage every 120 sec, simulating the rate of SF in patients with severe sleep apnea. For the non-sleep fragmentation (NSF) control mice, subjects were housed in SF chambers, but no sweeping bar movements occurred. The NSF groups matched collection times of ASF mice (1, 2, 6, 12, or 24 h; all groups, <em>n</em> = 10).  Both ASF and NSF groups were compared to a baseline group (time = 0) of mice collected at 0800 (<em>n</em> = 10). <strong> </strong> <em>Sample Collection. </em>After ASF or NSF treatments, mice were rapidly anesthetized using isoflurane induction (5%) and decapitated &lt;3 min of initial handling for tissue gene expression studies and blood collection for measurement of CORT and interleukin-6 (IL-6) levels (see below). Trunk blood was collected from mice, kept on ice for &lt;20 min, and spun at 3000×g for 30 min at 4°C. Serum was collected and stored at -80°C for corticosterone and IL-6 ELISA assays (see below). For gene expression analyses, three brain regions (prefrontal cortex (PFC), hypothalamus, and hippocampus), liver, spleen, heart, and epididymal white adipose tissue (EWAT) were dissected from mice and stored in RNAlater solution (ThermoScientific) in the freezer at -20°C. These particular brain regions and peripheral tissues were chosen because previous studies have demonstrated elevated pro-inflammatory gene expression from ASF. All tissue samples were stored at -20°C before RNA extraction.   <strong>Corticosterone and Interleukin-6 ELISA. </strong>Serum levels of corticosterone (n = 9-10/group) were measured using an ELISA kit (Catalogue number ADI-901-097, EnzoLife Sciences) which had a sensitivity of 26.99 pg/mL with cross-reactivity of &lt;30% deoxycorticosterone and &lt;2% progesterone. Samples were diluted 1:40 before running. The reaction was carried out in duplicate according to the kit instructions, and the average absorbance of the plate was determined using a plate reader (BioTek Synergy H1 Hybrid Reader). Average intra- and inter-assay variations were 2.85% and 2.65% respectively. IL-6 was measured in sera using ELISA MAX Deluxe kits (Catalogue number 431304; BioLegend, San Diego, CA). The assays were carried out according to the manufacturer's instructions, and the average intra- and interassay variations were 8.24% and 7.43% respectively. <strong>Cytokine gene expression.    </strong>RNA was extracted from liver, spleen, epidydimal white adipose tissue (EWAT), as well as the prefrontal cortex, hippocampus, and hypothalamus from brain using a RNeasy mini kit (Qiagen). RNA was extracted from the heart using a RNeasy Fibrous Tissue mini kit (Qiagen). All extractions were performed following the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific). Total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific, Cat number: 4368813) according to the manufacturer’s instructions and used as a template for determining relative cytokine gene expression using an ABI 7300 RTPCR system. Tissues were analyzed with cytokine primers/probes (IL1β: Mm00434228, TNFα: Mm00443258; ThermoFisher Scientific). Assay probes were labeled with florescent marker 5-FAM and quencher MGB at the 5’ end and 3’ end, respectively, and VIC-labeled 18S primer/probe (primer-limited; 4319413E; ThermoFisher Scientific) was used as an endogenous control. A multiplex PCR assay which included the genes of interest, and the endogenous control was run simultaneously for each sample. Samples were run in duplicate and the fold change in mRNA level was calculated as the relative mRNA expression levels, 2<sup>-ΔΔCt</sup>. The cycle threshold (Ct) at which the fluorescence exceeded background levels was used to calculate ΔCt (Ct[target gene] – Ct[18S]). Each Ct value was normalized against the highest Ct value of a control sample (ΔΔCt), and then the negative value of this power to 2 (2<sup> –ΔΔCt</sup>) was used for mRNA expression analysis.    
description: <strong> </strong> <strong>Statistical Analysis.
   </strong>Data are presented as mean (±SE). All
 statistical analyses were performed using GraphPad Prism (version 9.0).
 Two-way ANOVAs assessed the effect of sleep treatment (ASF or NSF), time
 (1h, 2h, 6h, 12h, 24h), and their interaction on mRNA expression of
 cytokines, serum CORT levels, and serum IL-6 concentration. One-way ANOVAs
 were used to assess whether ASF and NSF groups differed significantly from
 baseline levels (time 0h).  Tukey’s HSD and Bonferroni multiple
 comparisons were used for