{"paper_id":"01a5a156-ce4b-4dd9-987b-6e122e6d5f91","body_text":"Peripheral sympathectomy alters neuroinflammatory responses and microglial activity in response to sleep fragmentation in female mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Peripheral sympathectomy alters neuroinflammatory responses and microglial activity in response to sleep fragmentation in female mice Ila Mishra, Keelee B. Pullum, Kristen N. Eads, Anna R. Strunjas, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1336087/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Sleep loss, either induced by obstructive sleep apnea or other forms of sleep dysfunction, induces an inflammatory response, as commonly measured by increased circulating levels of pro-inflammatory cytokines. Increased catecholamines from sympathetic nervous system (SNS) activation regulates this peripheral inflammation. However, the role that catecholamines play in mediating neuroinflammation from sleep perturbations is undescribed. The aims of this study were to determine (i) the effect of peripheral SNS inhibition upon neuroinflammatory responses to sleep fragmentation (SF) and (ii) whether homeostasis can be restored after 1 week of recovery sleep. We measured gene expression levels of pro- and anti-inflammatory cytokines and microglial activity in brain (prefrontal cortex, hippocampus and hypothalamus) of female mice that were subjected to acute SF for 24 hours, chronic SF for 8 weeks, or 7 days of recovery after chronic SF. In each experiment, SF and control mice were peripherally sympathectomized with 6-OHDA (6-hydroxydopamine) or injected with vehicle. SF elevated cytokine mRNA expression in brain and increased microglial density and cell area in some regions. In addition, chronic SF promoted hyper-ramification in resting microglia upon exposure to chronic, but not acute, SF. Effects of chronic SF were more pronounced than acute SF, and 1 week of recovery was not sufficient to alleviate neuroinflammation. Importantly, 6-OHDA treatment significantly alleviated SF-induced inflammation and microglial responses. This study provides evidence of SNS regulation of neural inflammation from SF, suggesting a potential role for therapeutics that could mitigate neuroinflammatory responses to sleep dysfunction. 6-hydroxydopamine cytokines microglia neuroinflammation obstructive sleep apnea sleep fragmentation sympathetic nervous system Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background There is accumulating evidence that sleep dysfunction promotes inflammatory responses in brain and peripheral tissues, which can contribute to the development of chronic pathologies, such as metabolic and cardiovascular diseases and neurological disorders [ 1 – 3 ]. The global increase in obesity rates has led to a pronounced rise in the incidence of obstructive sleep apnea [ 4 ]. Chronic sleep fragmentation (SF) simulates the interrupted sleep experienced by patients with obstructive sleep apnea and other sleep disorders [ 5 – 7 ]. Concomitantly, sleep loss can increase autonomic sympathetic nervous system (SNS) activation, culminating in the release of norepinephrine (NE) from sympathetic nervous terminals, and NE and epinephrine (E) from the adrenal medullae. These catecholamines are immunomodulatory [ 3 ], and we have recently documented that pharmacological suppression of SNS ameliorates the inflammatory response in peripheral tissues of female mice exposed to acute and chronic sleep dysfunction [ 7 ]. However, the role that the SNS plays in mediating neuroinflammatory responses to sleep dysfunction is unexplored. Sleep loss-induced neurological damage and cognitive decline primarily involves microglia activation in the brain [ 8 ]. Microglia cells are resident phagocytes of the brain that act as sentinels of the nervous system to clean debris and regulate inflammatory responses to injury and infection [ 9 ]. Sleep deprivation can promote microglia activation in the absence of injury or infection [ 8 ], leading to release of pro-inflammatory cytokines and/or increased phagocytosis [ 10 ]. Microglia are activated in hippocampus after 5 days of sleep deprivation in rats [ 9 ], but acute sleep loss of 8 hours in mice is not sufficient to activate cortical microglia [ 8 ]. Thus, the temporal and special dynamics of microglia activity during sleep disturbances and deprivation are not fully elucidated. In the present study we examined the effects of acute and chronic SF on neural inflammatory responses in female mice. Females were chosen because of their general underrepresentation in neurobiological studies [ 11 ]. We also assessed whether 7 days of recovery sleep could sufficiently diminish neuroinflammation from chronic SF. To examine the effect of SNS activation on neuroinflammatory responses to SF, we peripherally sympathectomized mice using 6-hydroxydopamine (6-OHDA). We assessed pro-inflammatory cytokine gene expression and microglial morphology in three distinct brain regions (pre-frontal cortex, hippocampus and hypothalamus) that have been explored in previous studies using experimental sleep fragmentation [ 7 , 12 ]. Since peripheral signals of inflammation are proposed to affect neuroinflammation via the neural transmission of vagal afferents reacting to circulating pro-inflammatory agents or the pro-inflammatory agents themselves crossing the blood-brain barrier to directly affect microglia [ 13 ], we hypothesized that peripheral sympathectomy would alleviate the neuroinflammatory responses and diminish microglial activity to acute and chronic SF. Finally, we predicted that a 1-week recovery from chronic SF would reverse neuroinflammatory responses as was previously observed when assessing peripheral tissues [ 7 ]. Methods Animals and experimental protocol Female C57BL/6J mice between 8-12 weeks of age were used. Sleep fragmentation (SF) experiments were performed using automated SF chambers (Lafayette Instrument Company; Lafayette, IN; model 80390) with a thin layer of corn bedding as previously described [7]. These chambers ensure that mice are subjected to sleep fragmentation and not absolute sleep deprivation [12]. Mice were provided ad libitum access to water and food and housed in a 12:12-h light-dark photoperiod (lights on- 8am, 21°C ± 1°C) at Western Kentucky University. Subjects were acclimated to the SF chambers for 72 h prior to the commencement of sleep fragmentation experiments to diminish any carryover effects from the novel cage environment[14]. This study was conducted under the approval of the Institutional Animal Care and Use Committee at Western Kentucky University (#15-11), and procedures followed the National Institutes of Health’s “Guide for the Use and Care of Laboratory Animals” and ARRIVE guidelines. Acute Sleep Fragmentation (Acute SF) Female mice (n = 26) were chemically sympathectomized with a daily subcutaneous injection of 6-OHDA (0.1mg/g body mass/day) for 5 days while the remaining mice ( n =26) were injected with the same volume of vehicle (100 µl of 0.9% NaCl/day). Following the final injection on Day 5, experimental mice ( n = 26; n = 13 injected with vehicle and n = 13 injected with 6-OHDA) were subjected to acute SF i.e. a sweeping bar set to move horizontally every 120 seconds for 24 hrs, which simulates the rate of severe sleep apnea among humans ([15]. Controls ( n = 26; n = 13 injected with vehicle and n = 13 injected with 6-OHDA) received no sweeping bar movements. 6-OHDA treatment was effective in suppressing SNS activity because serum epinephrine levels were reduced in SF mice receiving 6-OHDA [7]. Chronic Sleep Fragmentation (Chronic SF) To induce chronic sleep fragmentation, mice ( n = 28) were subjected to a horizontal sweeping bar set to move every 120 seconds (30 swipes/h) during the light phase (i.e. from 8 am to 8 pm) every day for 8 weeks. To account for increased activity from daily chronic SF, control mice ( n = 26) were subjected to the same number of bar sweeps as experimental mice for only 3 hours of the light phase (i.e. from 8 am to 11am), albeit at 4 times the rate i.e. 2 swipes/minute, to control for daily activity induced by the bar movement. Mice were subcutaneously injected with either 6-OHDA (0.1mg/g body mass/day; n = 13 of SF mice, n = 13 of control mice) or vehicle (100 µl of 0.9% NaCl/day; n = 15 of SF mice, n = 13 of control mice) between 8-9 am for 5 consecutive days prior to termination of the experiment. Chronic Sleep Fragmentation + Recovery (Chronic SF + R) Mice were subjected to the chronic SF protocol above, but post SF, control ( n = 16) and SF mice ( n = 25) were subjected to a recovery period (no bar movement) for 7 days during which they were injected with either 6-OHDA (0.1mg/g body mass/day; n = 13 of chronic SF + R mice, n = 8 of control mice) or vehicle (100 µl of 0.9% NaCl/day; n = 12 of SF + R mice, n = 8 of control mice) for 5 days. For immunohistochemical studies, the experimental protocols mentioned above were followed, except that the chronic SF+ R experiment did not have a separate control group. Hence, in immunohistochemistry experiment 2, there were three sleep paradigms: controls, chronic SF and chronic SF+ R, each with vehicle or 6-OHDA treatment ( n = 5 per group except vehicle injected chronic SF+ recovery group with n = 4). Gene expression analysis In all three experiments, 24 hours following the final drug administration, brains of mice ( n = 8/ group except chronic SF + vehicle group of n = 10 mice) were sampled for RTPCR or immunohistochemistry analysis. Sample sizes of 10 were deemed necessary according to previous power analyses that demonstrate inherent variability in cytokine gene expression. For measurement of brain gene expression, brains were dissected from decapitated mice and stored in RNAlater solution (ThermoFischer Scientific) at 4°C until RNA extraction. RNA was extracted from hippocampus, pre-frontal cortex and hypothalamus using a RNeasy mini kit (Qiagen). RNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (ThermoFischer Scientific). Total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Life Technologies, Cat number: 4368813). The prepared cDNA was used as template for determining relative cytokine gene expression using an ABI 7300 RTPCR system. Cytokine probes ( IL1 β , IL-6 , TNFα , TGFβ ; Applied Biosystems) labelled with fluorescent marker 5-FAM at the 5’ end and quencher MGB at the 3’end were used for genes of interest along with 18S (primer-limited VIC-labelled probe) as the endogenous control according to the manufacturer’s instructions. Samples were run in duplicate and the fold change in mRNA levels was calculated as the relative mRNA expression levels, 2 -ΔΔCt [16]. Briefly, the cycle threshold (Ct) obtained by fluorescence exceeding background levels was used to calculate ΔCt(Ct[target gene] - Ct[18s]). Each Ct value was normalized against the lowest Ct value of a control sample. The negative value of this powered to 2 (2 -ΔΔCt ) was plotted. Immunohistochemistry For immunohistochemistry studies, 24 hours following final drug administration, mice were deeply anesthetized using isoflurane vapors and then transcardially perfused ( n = 5/group except recovery + vehicle group of n = 4) with ice-cold saline (0.9%) followed by 4% paraformaldehyde prepared in phosphate buffered saline (PBS). Brains were dissected out from perfused mice, post-fixed in the same fixative overnight, cryoprotected in 30% sucrose and stored at -80°C until cryosectioned. 30 µm sections from brains of control ( n = 5/vehicle or 6-OHDA treatment) and acute SF ( n = 5/vehicle or 6-OHDA treatment) mice from the first study, and control ( n = 5/vehicle or 6-OHDA treatment), chronic SF ( n = 5/vehicle or 6-OHDA treatment) and chronic SF+ R ( n = 4 injected with vehicle, n = 5 injected with 6-OHDA) mice from the second study were processed for immunohistochemistry of IBA-1. Sections were rinsed thrice in 10mM PBS, followed by an incubation in blocking solution (2.5% goat serum, 0.1% triton X) for 2 hours and overnight incubation (at 4°C) in the same blocking solution containing anti-Iba-1 (1:300, Catalogue number 019-19741, Wako Pure Chemicals Industries, China). Sections were then incubated with goat anti-rabbit secondary antibody (Alexa fluor 594, catalogue number A11037, Invitrogen, Life Technologies Corporation, Oregon, USA) for 2 hours, followed by washing of sections with 10mM phosphate buffer saline and mounting on slides. After 2 hours of air drying in a dark chamber, slides were mounted and permanently fixed with DAPI+antifade (Vectashield, H-1500, Vector Laboratories, Inc., CA 94010, USA). Image Analysis To quantify microglia, every sixth section of the brain was selected and immune-labelled with Iba1 antibody. Iba1 immunoreactivity (-ir) was visualized through a Zeiss Axioplan 2 epifluorescence microscope fitted with a Leica DFC300 FX digital camera. Omission of primary antibodies in control studies confirmed the absence of nonspecific immunoreactivity (data not shown). Iba-1-ir cells in captured images were manually counted by two operators blind to the experimental conditions using the cell counting plug-in in Image J (Version 1.45 J; National Institutes of Health, Bethesda, MD, USA). The brightness and contrast of images were adjusted to a firm setting for all sections to aid in visualization of immunostaining. A box of size 80000 µm 2 , 100000 µm 2 and 42000 µm 2 was overlaid on 100x (10x objective, 10x eyepiece) pictures of sections with cingulated cortex (Bregma: +0.74: +0.38), dentate gyrus (Bregma: -1.64: -2.12) and pre-optic area (Bregma: -0.94: -1.28), respectively, and region identification was based on the mouse brain atlas (Paxinos and Franklin, 2001; http://www.mbl.org/atlas170/atlas170_frame.html). Cells were counted in a total of 4 sections. Iba1-ir values were averaged from both sides of bilateral nuclei, and the count from 4 sections was summed for each mice. Total numbers of positive cells per animal were multiplied by six to estimate the number of cells per cingulate cortex, dentate gyrus and pre-optic area. For morphological analysis of microglia, all analysis were performed on maximum intensity projections (Z-project, maximum intensity function in Image J) of the Z-stacks collected with 0.7µm increments on a Olympus Flowview (FV1000) confocal microscope with 10X eyepiece and 100X objective (1000X). Microglia were classified as ramified (numerous thin processes, radial branching), primed (thickened processes, increased polarity, and proliferation with reduced secondary branching), reactive (thickened stout processes with highly reduced branching), or amoeboid (rounded soma with no branching; Figure 1A–D) based on standard morphological criteria [17]. Sholl analysis was performed over 20 concentric circles, with the inner radius of 6µm and outer radius of 35µm on a total of 15 ramified microglia (5 from cingulated cortex + 5 from dentate gyrus + 5 from pre-optic area) per brain and 5 brain/group. Analysis was done by 3 operators blind to the experimental conditions. Non-overlapping microglia cells that were in a ramified state exhibiting intact microglial processes unobscured by either background labelling or other cells were photographed and analysed. Statistical Analysis Data are presented as mean (±SE). All statistical analyses were done using GraphPad prism (version 6.0), IBM SPSS Statistics version 20 software as appropriate. Two-way ANOVAs assessed the effect of sleep fragmentation (factor 1), 6-OHDA treatment (factor 2) and their interaction on mRNA expression of cytokines and Iba-1 immunohistochemistry in brain. A three-factor analysis, using Univariate General linear model (GLM) tested the effects of sleep fragmentation (factor 1), 6-OHDA treatment (factor 2), and distance from soma (factor 3), and their interactions on morphology of the Iba-1 staining. Bonferroni multiple comparisons were used for posthoc analysis. p < 0.05 was considered statistically significant. Results SF-induced effects on cytokine mRNA expression levels in brain There was a significant effect of acute SF on IL-1β and IL-6 expression levels in prefrontal cortex ( IL-1β : F 1,28 = 4.69, p = 0.039, IL-6 : F 1,28 = 8.07, p = 0.008, 2-way ANOVA), while acute SF induced effects on IL-1β expression levels in both prefrontal cortex and hippocampus were dependent on 6-OHDA treatment (prefrontal cortex: interaction- F 1,28 = 7.81, p = 0.009, hippocampus: interaction - F 1,26 = 6.52, p = 0.0169, 2-way ANOVA). Similarly, in hypothalamus, there was a significant effect of acute SF on IL-1β ( F 1,28 = 6.07, p = 0.020), and an interaction of acute SF and 6-OHDA on IL-6 ( F 1,28 = 16.21, p = 0.0004; 2-way ANOVA). TNFα levels were unaffected by the experimental treatment in brain tissues while TGFβ levels in all three brain regions were significantly affected by acute SF (prefrontal cortex: F 1,28 = 8.62, p = 0.007; hippocampus: F 1,28 = 14.29, p = 0.0008; hypothalamus: F 1,28 = 15.07, p = 0.0006) and 6-OHDA treatment (except in prefrontal cortex; hippocampus: F 1,28 = 14.77, p = 0.0006, hypothalamus: F 1,28 = 10.55, p = 0.0030; 2-way ANOVA). There was a significant effect of chronic SF on the expression levels of all genes assessed in prefrontal cortex (except TNFα ; IL-1β : F 1,30 = 4.29, p = 0.047; IL-6 : F 1,29 = 7.512, p = 0.0106; TGFβ : F 1,29 = 6.510, p = 0.0163) along with a significant effect of 6-OHDA treatment and its interaction with chronic SF on TNFα and IL-1β , respectively ( TNFα : 6-OHDA- F 1,31 = 4.94, p = 0.034; IL-1β : interaction- F 1,30 = 4.498, p = 0.042). Further, hippocampal TGFβ expression was significantly affected by 6-OHDA ( F 1,30 = 4.43, p = 0.044) and its interaction with chronic SF ( F 1,30 = 6.17, p = 0.019; 2-way ANOVA). Similarly, hypothalamic IL-1β expression was significantly affected by 6-OHDA treatment ( F 1,29 = 19.11, p = 0.0002), and hypothalamic TNFα levels were significantly affected by chronic SF ( F 1,29 = 8.27, p = 0.0076) and 6-OHDA ( F 1,29 = 28.16, p < 0.0001; 2-way ANOVA). One week of recovery sleep significantly affected IL-1β expression in hippocampus ( F 1,28 = 5.97, p = 0.021) and hypothalamus ( F 1,28 = 5.13, p = 0.032), along with a significant effect of recovery and 6-OHDA treatment on TNFα expression in brain (hippocampus: recovery - F 1,28 = 51.91, p < 0.0001, 6-OHDA- F 1,28 = 6.49, p = 0.0166; hypothalamus: recovery - F 1,28 = 4.36, p = 0.046, 6-OHDA- F 1,28 = 4.84, p = 0.036). IL-6 expression levels in prefrontal cortex and hippocampus were significantly affected by recovery (prefrontal cortex: F 1,28 = 14.38, p = 0.0007; hippocampus: F 1,28 = 7.97, p = 0.0087) and 6-OHDA treatment (prefrontal cortex: F 1,28 = 14.98, p = 0.0006; hippocampus: F 1,28 = 23.54, p < 0.0001), the expression levels in hypothalamus were significantly affected by 6-OHDA ( F 1,28 = 42.39, p < 0.0001) and its interaction with recovery ( F 1,28 = 10.30, p = 0.0034). Neural TGFβ expression levels were significantly affected by recovery (hippocampus: F 1,28 = 11.00, p = 0.003; hypothalamus: F 1,28 = 11.37, p = 0.002), 6-OHDA (prefrontal cortex: F 1,28 = 5.56, p = 0.026; hippocampus: F 1,28 = 12.75, p = 0.001; hypothalamus: F 1,28 = 14.48, p = 0.0007) and their interaction (prefrontal cortex: F 1,28 = 12.68, p = 0.0013; hypothalamus: F 1,28 = 13.81, p = 0.0009), except the effect of recovery and its interaction with 6-OHDA in prefrontal cortex and hippocampus, respectively (2-way ANOVA). In comparison to controls, IL-1β expression under both SF regimes was significantly increased in brain regions (except hippocampus of chronic SF exp; Bonferroni posthoc test, p < 0.05, Fig. 2a, e, i, 3a, i). Acute SF caused a significant reduction in expression levels of TGFβ in all three brain tissues, and in IL-6 in prefrontal cortex and hypothalamus (Bonferroni posthoc test, p < 0.05, Fig. 2b, d, f, h, j, l), as opposed to chronic SF which lead to a significant increase in IL-6 expression in prefrontal cortex, TNFα in hypothalamus and TGFβ in hippocampus (Bonferroni posthoc test, p < 0.05, Fig. 3b, h, k). A recovery period of 1 week after chronic SF was enough to negate the effects of chronic SF (except IL-6 in prefrontal cortex). In fact, IL-6 , TNFα and TGFβ expression in hippocampus and hypothalamus and IL-1β expression in hypothalamus was significantly lower in recovery mice compared with controls (Bonferroni posthoc comparison, p < 0.05, Fig 4f-l). In addition, lack of significant differences between 6-OHDA administered controls and experimental groups in all experiments (except hypothalamic TNFα of chronic SF+R experiment), along with significant reduction in hypothalamic IL-1β and TNFα in 6-OHDA-treated chronic SF mice, and in TGFβ in hippocampus and cortex of 6-OHDA treated acute and chronic SF mice, indicated that chemical sympathectomy significantly attenuated SF-induced neuroinflammatory responses (Bonferroni posthoc test, p < 0.05, fig. 2-4). SF-induced effects on microglia activity Iba-ir cell number was significantly affected by acute SF (cortex: F 1,16 = 15.60, p = 0.001; hippocampus: F 1,16 = 14.79, p = 0.001; pre-optic area: F 1,16 = 6.03, p = 0.026), 6-OHDA treatment (cortex: F 1,16 = 15.60, p = 0.001; hippocampus: F 1,16 = 16.33, p = 0.001), and their interaction (cortex: F 1,16 = 26.49, p < 0.0001; hippocampus: F 1,16 = 10.45, p = 0.005; pre-optic area: F 1,16 = 5.56, p = 0.031) in all three brain regions, except an effect of 6-OHDA in pre-optic area, as determined by 2-way ANOVA. In all three brain regions, acute SF significantly increased Iba-ir, which was significantly decreased with 6-OHDA treatment (Bonferroni posthoc test, p < 0.05, Fig. 5a, e, i). Similarly, Iba-ir soma area was significantly affected by acute SF in pre-optic area ( F 1,96 = 6.46, p = 0.012), by 6-OHDA treatment in cortex ( F 1,96 = 12.04, p = 0.0008), and by their interaction in hippocampus ( F 1,96 = 4.87, p = 0.029) and pre-optic area ( F 1,96 = 4.07, p = 0.047; 2-way ANOVA), with significantly greater –ir soma area in pre-optic area of mice subjected to acute SF compared with controls (Bonferroni posthoc test, p < 0.05, Fig. 5b, f, j). Iba-ir soma area in cortex of acute SF mice was significantly greater than their counterparts pre-treated with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig. 5b). In the second experiment, there was a significant effect of SF on Iba-ir cell number in cortex ( F 1,23 = 26.24, p < 0.0001) and hippocampus ( F 1,23 = 4.43, p = 0.023), of 6-OHDA treatment and its interaction with SF in cortex (6-OHDA: F 1,23 = 8.10, p = 0.009; SF x 6-OHDA: F 2,23 = 19.30, p < 0.0001; 2-way ANOVA). Interestingly, Iba-ir cell number in cortex and hippocampus of mice subjected to chronic SF > recovery > controls (Bonferroni posthoc test, p < 0.05 Fig. 6a, e). Furthermore, 6-OHDA treatment significantly reduced Iba-ir in mice subjected to chronic SF (Bonferroni posthoc test, p < 0.05; Fig. 6a, e, i). There was a significant effect of SF on Iba-ir soma area in cortex ( F 2,139 = 6.49, p = 0.002) and hippocampus ( F 2,139 = 4.68, p = 0.011), and of 6-OHDA treatment ( F 1,139 = 12.51, p = 0.0006) and its interaction with SF ( F 2,139 = 6.73, p = 0.002) on Iba-ir soma area in pre-optic area (2-way ANOVA). Iba-ir soma area was significantly increased in chronic SF mice compared with recovery and controls (except in hippocampus; Bonferroni posthoc test, p < 0.05, Fig. 6b, f, j). Further, chronic SF mice had significantly lower Iba-ir soma area in pre-optic area when treated with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig. 6j). Three-factor univariate general linear models indicated a significant effect of acute SF and 6-OHDA treatment on microglial ramification in ( F 1,1824 = 19.66, p < 0.0001) and pre-optic area ( F 1,1824 = 43.31, p < 0.0001). Microglia ramification significantly varied with distance from soma (cortex: F 18,1824 = 38.38, p < 0.0001; hippocampus: F 18,1824 = 20.97, p < 0.0001; pre-optic area: F 18,1824 = 10.02, p < 0.0001) and acute SF induced effects were dependent on 6-OHDA treatment in all three regions (acute SF x 6-OHDA interaction - cortex: F 1,1824 = 6.124, p = 0.013; hippocampus: F 1,1824 = 25.76, p < 0.0001; pre-optic area: F 1,1824 = 15.99, p < 0.0001). There was a significant three-way interaction only in pre-optic area (acute SF x 6-OHDA x distance from soma: F 18,1824 = 3.38, p < 0.0001, univariate GLM). In cortex, acute SF significantly increased the ramification at 6.5 µm from the centre of soma when compared with controls (Bonferroni posthoc test, p < 0.05, Fig 5c). 6-OHDA significantly affected the microglia ramification in controls, with higher ramification in cortex (between 9 to 16 µm from the soma centre), hippocampus (23 µm from the soma centre) and pre-optic area (at 26 and 29 µm from soma centre; Bonferroni posthoc test, p < 0.05, Fig 5c, d, g, h, k, l). Similarly, acute SF mice when injected with 6-OHDA, showed higher ramification in hippocampus at a distance of 29-30.5 µm from the centre of soma (Bonferroni posthoc test, p < 0.05, Fig 5g, h). Chronic SF had more profound effects on microglia morphology with a significant effect of (cortex: F 2,2754 = 108.79, p < 0.0001; hippocampus: F 2,2754 = 24.59, p < 0.0001; pre-optic area: F 2,2754 = 72.93, p < 0.0001), 6-OHDA treatment (cortex: F 1,2754 = 5.14, p = 0.023; hippocampus: F 1,2754 = 16.51, p < 0.0001; pre-optic area: F 1,2754 = 113.17, p < 0.0001), distance from soma (cortex: F 18,2754 = 40.82, p < 0.0001; hippocampus: F 18,2754 = 37.55, p < 0.0001; pre-optic area: F 18,2754 = 29.74, p < 0.0001), and SF x 6-OHDA interaction (cortex: F 2,2754 = 52.79, p < 0.0001; hippocampus: F 2,2754 = 37.78, p < 0.0001; pre-optic area: F 2,2754 = 28.68, p < 0.0001) in all three brain regions, as determined by univariate GLM. In addition, there was a significant interaction effect of distance from soma with SF ( F 36,2754 = 1.83, p = 0.001) and 6-OHDA treatment ( F 18,2754 = 2.65, p < 0.0001) in cortex and pre-optic area, respectively. There was a significant three-way interaction in hippocampus ( F 36,2754 = 1.91, p = 0.001) and pre-optic area ( F 36,2754 = 2.16, p < 0.0001), but not in cortex. In comparison with controls, chronic SF caused microglia hyper-ramification in all three brain regions (distance from centre of soma: 14 to 32 µm in cortex, 17 to 26 µm in hippocampus, and 20 to 35 µm in pre-optic area). A significantly higher ramification in cortex at 11 to 32 µm, in hippocampus at 26 to 27.5 and 32 to 33.5 µm, and in pre-optic area at 20 to 35 µm from centre of soma was observed in chronic SF mice compared with recovery mice (Bonferroni posthoc test, p < 0.05, Fig 6c, d, g, h, k, l). Hippocampal microglial response was still observed after 1 week of recovery, as ramification was significantly higher in mice subjected to recovery at 12.5 to 14 µm distance from centre of soma (Bonferroni posthoc test, p < 0.05, Fig 6g, h). Further, 6-OHDA administration diminished the neuroinflammatory response from chronic SF, as evidenced by significantly lower microglia ramification in cortex (at 23 to 33.5 µm distance from centre of soma), hippocampus (at 17 to 23 µm, 26 and 30.5 µm distance from centre of soma) and pre-optic area (at 14, and 20 to 30.5 µm distance from centre of soma) in mice subjected to 6-OHDA treatment following chronic SF, relative to their counterparts injected with vehicle (Bonferroni posthoc test, p < 0.05, Fig 6c, d, g, h, k, l). The pre-optic area of recovery mice injected with 6-OHDA had lower microglia ramification at 11 to 14 µm distance from centre of soma, compared with recovery mice not injected with 6-OHDA (Bonferroni posthoc test, p < 0.05, Fig 6k, l). Discussion Sleep disturbances can promote neuroinflammation and blood-brain barrier disruption mediated by inflammatory molecules produced from astrocytes and microglia. For instance, sleep restriction for 72 h elevated IL-1β , IL-6 and TNFα in hippocampus and basal forebrain of rats [ 18 ] and chronic sleep restriction for 21 days elevated IL-6 and TNFα in hippocampus and TNFα in prefrontal cortex of rats [ 19 ]. Similarly, in the present study, sleep fragmentation (SF) promoted neuroinflammatory responses albeit in a brain region-specific and SF-duration dependent manner. In addition to having proinflammatory roles, elevated IL-1β in brain following acute and chronic SF (except in hippocampus), and TNFα in hypothalamus following chronic SF may indicate an accumulation of SRS (sleep regulatory substances) following a period of prolonged wakefulness [ 20 ]. The increase in neural TNFα response after chronic but not acute SF is in corroboration with previous reports [ 12 , 21 , 22 ]. An induction of an anti-inflammatory environment in the brain after long-term SF has been reported before [ 21 , 23 ]. Increased hippocampal TGFβ expression following chronic SF perhaps provided evidence for neuroprotection from inflammation. Further, while primarily pro-inflammatory, IL-6 has been described to play anti-inflammatory roles in some scenarios, causing stimulation of IL-10 synthesis [ 24 ] and inhibition of IL-1 and TNFα production [ 25 ]. While the precise functioning of IL-6 is not deciphered in the present study, opposite trends in its mRNA expression under acute and chronic SF seems to suggest an effort to closely regulate neuroinflammatory response to SF. Microglial activation is considered a main source of neuroinflammation among other factors [ 9 ]. Sleep loss has been shown to affect microglial morphology[ 26 ]. In mature CNS, microglia are in a ‘resting state’ with highly ramified and motile processes, continuously surveying the surrounding environment. In response to injury, microglia shift from resting to a primed phagocytic ‘activated’ state [ 26 ]. Sleep loss, in the absence of injury, has been reported to increase phagocytic activity of microglia [ 8 ]. On the other hand, psychological and mechanical stress has also been shown to increase the microglial secondary branching in the cortex of rats [ 27 , 28 ], thus suggesting that microglial activation in response to homeostatic perturbations can range from mild non-inflammatory hyper-ramified phenotype to pro-inflammatory, apoptotic and phagocytic phenotypes [ 26 ]. Unlike chronic OSA and SF, neither OSA nor sleep deprivation/restriction of 1 night has been previously reported to increase the phagocytic activity of microglia [ 8 , 29 ]. Therefore, we surmise that perhaps acute SF of 1 night might affect the mild ‘non-inflammatory’ instead of the ‘activated’ phagocytic morphology of microglia. Absence of microglial hyper-ramification in acute SF mice suggested that SF-induced neuroinflammatory changes may not necessarily be associated with changes in microglial morphology [ 26 ]. On the other hand, increased ramification after chronic SF suggests that chronic sleep perturbations induced microglial morphological changes that may include an increase in ‘activated’ phenotype involved in enhanced phagocytosis of synaptic elements [ 8 ] and an increased ramification of the ‘resting’ phenotype possibly involved in increased surveillance and scanning of microenvironment [ 27 ]. Given the reversibility and complexity of microglia morphology, especially across brain regions [ 30 ], we analysed only the ramified morphology of microglia. SF-induced alterations in the phagocytic phenotype need to be tested in future studies, and thus presently cannot be ruled out. Usually, inflammation from sleep loss return to basal levels after sleep recovery. However, depending on duration of sleep loss and recovery, some immune components may remain altered after sleep recovery [ 31 ]. In this study, while seven days of recovery completely alleviates inflammatory responses in peripheral tissues from a previous study [ 7 ], it was not sufficient to restore homeostasis in brain. Elevated Iba-ir in all three brain regions and increased IL-6 expression in cortex of recovery mice indicated that neuroinflammatory responses persisted at least 1 week following chronic SF. Consistent with our finding, a recent study reported that even after three weeks of recovery from sleep loss, neuronal apoptosis, microglial activation and IL-6 response still occurred in hippocampus of mice [ 32 ]. Furthermore, similar to our results, anti-inflammatory responses lower than baseline levels have been reported in mice subjected to 20 days of recovery post SD of 192 hours [ 21 ]. In addition, we documented a significant reduction in cytokine gene expression in brain after 7 days of recovery sleep. Perhaps, these changes may be a compensatory response that limits neurological and cardiovascular dysfunction related to SF-induced hyperactivation of inflammation over prolonged periods of SF. Irrespective of direction of deviation from baseline, it is plausible that one possible mediator of delayed return of neuroinflammatory to baseline homeostatic state was SNS and not the hypothalamic-pituitary-adrenal axis, since high levels of NE were found after 1 week of recovery sleep [ 7 ]. Our initial hypothesis that the SNS contributes to neuroinflammatory responses to sleep loss has been supported, as chemical sympathectomy significantly altered the inflammatory responses in all three SF paradigms examined. These results reiterate the complexities of potential NEI (neuro-endocrine-immune) interactions by which SF can affect immune functioning [ 31 , 33 ]. Peripheral sympathectomy also successfully alleviated SF-induced neuroinflammatory responses. Perhaps one or more of the following established mechanisms contributed to these results: (1) a passive diffusion of peripheral cytokines to brain via circumventricular organs or an active transport with carrier proteins across blood brain barrier (BBB) affected the microglial activity and neuroinflammatory processes (2) peripheral signals stimulated the endothelial cells of BBB to secrete molecules affecting activity of neurons and glia and/or (3) the autonomic nervous system itself via vagal afferents activated the neuroinflammatory responses [ 10 ]. Interestingly, 6-OHDA treatment also altered baseline cytokine levels. More specifically, TGFβ in hippocampus and IL-6 in hypothalamus was significantly lowered, while IL-1β in hippocampus was significantly increased in control mice after chemical sympathectomy in acute SF experiment. IL-6 , TNFα and TGFβ in hypothalamus and hippocampus (except TNFα ) of control mice were significantly attenuated after 6-OHDA treatment in the recovery experiment. The effects of chemical sympathectomy on baseline immune function in chronic SF experiment were less profound with only a significant reduction in hypothalamic TNFα expression in controls treated with 6-OHDA. Such differences between the three experiments could be attributed to the differences in experimental paradigms and duration of treatments involved (cf. Figure 1 ). Lastly, our data suggests that baseline neuroinflammatory responses of hypothalamus and hippocampus were more susceptible to peripheral 6-OHDA treatment than the cortex. Further studies are warranted to understand how peripheral signals activate neuroinflammation across different brain regions. Historically, the bias towards use of male rodents in research has resulted in data gaps to effectively discern the effects of sleep abnormalities upon female physiology. Given the sexual differences in susceptibility to diseases including neurological and immune diseases [ 34 , 35 ], this study provides critical insight regarding effects of sleep disruption on female peripheral and neural immune responses. Sex steroids regulate the development and maturation of immune cells and responses [ 35 ]. As result, inflammatory profiles [ 34 , 36 ] and microglial activity [ 36 ] differ between the sexes. Therefore, caution is warranted before extrapolating these results to responses in males. Paradoxical SD affects estrous cyclicity in rats [ 37 ]. Since we the reproductive status was not tested in mice of our study, the involvement of reproductive hormones in regulating SF-induced inflammation and microglial responses cannot be ruled out. Restraint stress also affects phasing of the estrous cycle in female rats, although there is large individual variation [ 38 ]. Because sample sizes in our study were small, future studies should evalute the effect of estrous cycling upon SF-induced inflammation using a greater sample size. Conclusions This study systematically evaluated the SF-associated neuroinflammation and microglial responses of female mice. Changes in inflammatory responses seemed to be reflective of stress-axes activation and were related to the duration of SF [ 7 ]. Unlike previous reports, our experimental approach of exposing mice to 8 weeks of SF can more accurately be expected to mimic sleep abnormalities, such as OSA and the diseases associated with it, in the modern world. Moreover, it is important to emphasize that a recovery sleep of seven nights following such chronic sleep fragmentation was clearly insufficient for a full return to homeostasis. Importantly, to our knowledge this is the first study showing chronic SF-induced subtle changes in the morphology of microglia primarily characterised as ‘resting’. Finally, this study provides evidence of a critical contribution of SNS in development of an inflammatory state. These findings could lead to novel therapeutic interventions that target the SNS for treating inflammation-dependent disorders, such as cardiovascular diseases. Declarations Acknowledgments We thank Melanie Richter and Nicholas Wheeler for sampling, and Naomi Rowland for assistance with RTPCR. This research was supported by the National Institutes of Health (R15GM117534) and the Kentucky Biomedical Research Infrastructure Network (NIGMS grant #8P20GM103436-14) to NTA. Author contributions IM wrote the primary draft, performed the experiments and primary analysis, KBP, KE and AS analysed the data, and NTA designed the study, wrote the final draft and acquired funding. All authors read and approved the final manuscript. Data availability The gene expression and image analysis datasets generated for this study are available from Figshare (https://doi.org/10.6084/m9.figshare.19369592.v1). Raw image files are available from the corresponding author on reasonable request due to large file sizes. Competing interests The authors declare that they have no competing interests. References Rajaratnam, S. M. & Arendt, J. Health in a 24-h society. Lancet 358 , 999–1005 (2001). Schwartz S., et al . Insomnia and heart disease A review of epidemiologic studies. J. Psychosom. Res. 47 , 313–33 (1999). Mullington, J. M., Haack, M., Toth, M., Serrador, J. M., & Meier-Ewert, H. K. Cardiovascular, inflammatory, and metabolic consequences of sleep deprivation. Prog. Cardiovasc. Dis. 51 , 294–302 (2009). Ardissino, M., et al . Sleep disordered breathing, obesity and atrial fibrillation: a mendelian randomisation study. Genes 13 , 104 (2022). 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Roles of microglial phagocytosis and inflammatory mediators in the pathophysiology of sleep disorders. Front. Cell. Neurosci. 11 , 250 (2017). Beery, A. K. Inclusion of females does not increase variability in rodent research studies. Curr. Opin. Behav. Sci. 23 , 143–9 (2018). Dumaine, J. E. & Ashley, N. T. Acute sleep fragmentation induces tissue-specific changes in cytokine gene expression and increases serum corticosterone concentration. Am. J. Phys. Regul. Integr. Comp. Physiol. 308 , R1062–9 (2015). Unnikrishnan, D., Jun, J., & Polotsky, V. Inflammation in sleep apnea: An update. Rev. Endocr. Metab. Disord. 16 , 25–34 (2015). Ashley, N. T., Sams, D. W., Brown, A. C., Dumaine, J. E. Novel environment influences the effect of paradoxical sleep deprivation upon brain and peripheral cytokine gene expression. Neurosci. Lett. 615 , 55–9 (2016). Goyal, M. & Johnson, J. Obstructive Sleep Apnea Diagnosis and Management. Mo. Med. 114 , 120–4 (2017). Livak KJ & Schmittgen, T. D. 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Chronic stress induced remodeling of the prefrontal cortex: structural re-organization of microglia and the inhibitory effect of minocycline. Cereb. Cortex. 23 , 1784–97 (2013). Streit, W. J., Walter, S. A. & Pennell, N. A. Reactive microgliosis. Prog Neurobiol. 57 , 563–81 (1999). Sapin, E. et al. Chronic intermittent hypoxia induces chronic low-grade neuroinflammation in the dorsal hippocampus of mice. Sleep 38 , 1537–46 (2015). Del Mar Fernández-Arjona, M., Grondona, J. M., Granados-Durán, P., Fernández-Llebrez, P. & López-Ávalos, M. D. Microglia morphological categorization in a rat model of neuroinflammation by hierarchical cluster and principal components analysis. Front. Cell Neurosci. 11 , 235 (2017). Faraut, B., Boudjeltia, K. Z., Vanhamme, L. & Kerkhofs, M. Immune, inflammatory and cardiovascular consequences of sleep restriction and recovery. Sleep Med. Rev. 16 , 137–49 (2012). Yin, M. et al. Assessment of mouse cognitive and anxiety-like behaviors and hippocampal inflammation following a repeated and intermittent paradoxical sleep deprivation procedure. Behav. Brain Res. 321 , 69–78 (2017). Ashley, N. T. & Demas, G.E. Neuroendocrine-immune circuits, phenotypes, and interactions. Horm. Behav. 87 , 25–34 (2017). Villa, A. et al. Sex-specific features of microglia from adult mice. Cell Rep. 23 , 3501–11 (2018). van Leeuwen, W. M. A. et al. Sleep restriction increases the risk of developing cardiovascular diseases by augmenting proinflammatory responses through IL-17 and CRP. Plos One 4 , e4589 (2009). Guneykaya, D. et al. Transcriptional and translational differences of microglia from male and female brains. Cell Rep. 24 , 2773–2783.e6 (2018). Antunes, I. B., Andersen, M. L., Baracat, E. C. & Tufik, S. The effects of paradoxical sleep deprivation on estrous cycles of the female rats. Horm. Behav. 49 , 433–40 (2006). Bollinger, J. L., Burns, C. M. B. & Wellman, C. L. Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex. Brain Behav. Immun. 52 , 88–97 (2016). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-1336087\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Article\",\"associatedPublications\":[],\"authors\":[{\"id\":94460282,\"identity\":\"a2e2881f-68a7-4ff7-858a-9959e39276fc\",\"order_by\":0,\"name\":\"Ila Mishra\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Case Western Reserve University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Ila\",\"middleName\":\"\",\"lastName\":\"Mishra\",\"suffix\":\"\"},{\"id\":94460283,\"identity\":\"fc3b9ef4-0e22-481a-8323-55748a41e32a\",\"order_by\":1,\"name\":\"Keelee B. Pullum\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"University of Pennsylvania\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Keelee\",\"middleName\":\"B.\",\"lastName\":\"Pullum\",\"suffix\":\"\"},{\"id\":94460284,\"identity\":\"406cb817-c4e4-4047-a06a-b4c81875010b\",\"order_by\":2,\"name\":\"Kristen N. Eads\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Western Kentucky University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Kristen\",\"middleName\":\"N.\",\"lastName\":\"Eads\",\"suffix\":\"\"},{\"id\":94460285,\"identity\":\"45298f30-c8ac-4401-83ba-15ea6c225dd3\",\"order_by\":3,\"name\":\"Anna R. Strunjas\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Western Kentucky University\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Anna\",\"middleName\":\"R.\",\"lastName\":\"Strunjas\",\"suffix\":\"\"},{\"id\":94460286,\"identity\":\"5ff250cb-7266-4d67-b873-dcceedc413e1\",\"order_by\":4,\"name\":\"Noah T. Ashley\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYHACNhCRwMfAwPiA4QCYTaQWIMlsQLIWNgmitJizNz97wFBTl8fG3mNWzXPmMAM/e44BXi2WPcfMDRiOHS5m4zljdpvnxmEGyZ43+LUY3Egwk2BgO5DYJpFjdjvnw2GgCAFbDG6kf5Ng+FeX2Cb/xqwYpMWesJYcMwnGNmagLTxmzDlAhxlIENJy5kyZRGLf4cQ2nrRi6T9n0nkkzjwrwK/lePs2iQ/f6hL72Q9v/DjjmLUcf3vyBrxawCABTHKA3cNDWDkCsD8gRfUoGAWjYBSMIAAAMSFHoQmFf88AAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Western Kentucky University\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Noah\",\"middleName\":\"T.\",\"lastName\":\"Ashley\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2022-02-07 17:44:04\",\"currentVersionCode\":2,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-1336087/v2\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-1336087/v2\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":19788285,\"identity\":\"3b738a33-a579-44cf-88c5-e93c67d46a8f\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:10:30\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":287558,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eExperimental protocol\\u003c/em\\u003e. \\u003c/strong\\u003eThree experiments were performed with experimental mice subjected to sleep fragmentation (SF) in an automated sleep fragmentation chamber for 24 hr (experiment 1: Acute SF), 8 weeks (experiment 2: Chronic SF), or 8 weeks of chronic SF followed by undisturbed sleep of 1 week (experiment 3: Chronic SF + recovery). A horizontally moving automated sweeping bar set to swipe across the chamber every 2 minutes ensured experimental group mice were sleep fragmented. Controls in chronic SF and recovery experiment were also subjected to a sweeping bar, although at a higher speed, and for lesser duration (2 swipes/minute for 3 hours aligned with lights on) to control for the increased activity induced effects between groups. During the recovery phase, mice of both- control and experimental groups were not subjected to sweeping bar swipes. On last 5 days of the experiments, mice were injected subcutaneously with 0.9% saline or 6-OHDA (6-hydroxydopamine). After 24 hours of final injection, mice were either decapitated for tissue gene expression study or transcardially perfused for immunohistochemical study. All mice were \\u0026gt; 8 weeks of age and were subjected to 12h light: 12h dark cycles with lights on at 8am and Lights off at 8pm, and provided food and water \\u003cem\\u003ead libitum\\u003c/em\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/557ff90b59defb1df3556557.png\"},{\"id\":19789820,\"identity\":\"0141845d-7a8e-445c-ae23-8a08ee6c6ac0\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:15:30\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":119289,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAcute Sleep fragmentation altered cytokine mRNA expression levels in brain tissues. \\u003c/em\\u003e\\u003c/strong\\u003eMean (± SE) gene expression levels of \\u003cem\\u003eIL-1\\u003c/em\\u003e, \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNFα\\u003c/em\\u003e, and \\u003cem\\u003eTGFβ\\u003c/em\\u003e in Prefrontal cortex (a-d), hippocampus (e-h), and hypothalamus (i-l) of vehicle-injected or chemically sympathectomized mice subjected to control or acute SF (n = 8/group). Asterisk (*) and pound (#) indicate a significant effect of SF and chemical sympathectomy, respectively, as determined by Bonferroni posthoc test followed by 2-way ANOVA. For statistical significance, alpha (α) was set at 0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/0a1e86f8bd07b2b8639886ea.png\"},{\"id\":19788286,\"identity\":\"64265ca9-18a6-498e-a2e0-ced0289c2a4f\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:10:30\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":76032,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eChronic Sleep fragmentation altered cytokine mRNA expression levels in brain tissues. \\u003c/em\\u003e\\u003c/strong\\u003eMean (± SE) gene expression levels of \\u003cem\\u003eIL-1\\u003c/em\\u003e, \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNFα\\u003c/em\\u003e, and \\u003cem\\u003eTGFβ\\u003c/em\\u003e in Prefrontal cortex (a-d), hippocampus (e-h), and hypothalamus (i-l) of vehicle-injected or chemically sympathectomized mice subjected to control or chronic SF (n = 8/group except for chronic SF + vehicle group of n = 10 mice). Asterisk (*) and pound (#) indicate a significant effect of SF and chemical sympathectomy, respectively, as determined by Bonferroni posthoc test followed by 2-way ANOVA. For statistical significance, alpha (α) was set at 0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/1e4840b589dd1a7e47660884.png\"},{\"id\":19788289,\"identity\":\"fa83ae85-09fe-46a4-a717-d8e1b6b565fd\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:10:30\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":78451,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eOne week of recovery altered cytokine mRNA expression levels in brain tissues. \\u003c/em\\u003e\\u003c/strong\\u003eMean (± SE) gene expression levels of \\u003cem\\u003eIL-1\\u003c/em\\u003e, \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNFα\\u003c/em\\u003e, and \\u003cem\\u003eTGFβ\\u003c/em\\u003e in Prefrontal cortex (a-d), hippocampus (e-h), and hypothalamus (i-l) of vehicle-injected or chemically sympathectomized mice subjected to control or one week recovery following chronic SF (n = 8/group). Asterisk (*) and pound (#) indicate a significant effect of SF and chemical sympathectomy, respectively, as determined by Bonferroni posthoc test followed by 2-way ANOVA. For statistical significance, alpha (α) was set at 0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/f227924377f7a64a65be0392.png\"},{\"id\":19789819,\"identity\":\"b9057d31-0643-42b2-9f66-7f83ec67f9ec\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:15:30\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":757086,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eAcute sleep fragmentation altered microglia activation. \\u003c/em\\u003e\\u003c/strong\\u003eMean (± SE) Iba-1-ir cell number (a, e, i), cell area (b, f, j), and ramification as determined by sholl analysis (c-d, g-h, k-l) in cortex (cingulated cortex; upper panel), hippocampus (dentate gyrus; middle panel) and hypothalamus (pre-optic area; lower panel) of vehicle-injected or chemically sympathectomized mice subjected to control or acute SF (n = 5/group). The magnified view in each micrograph represents ramified microglia (magnification: 1000x) subjected to sholl analysis. Asterisk (*) and pound (#) indicate a significant effect of SF and chemical sympathectomy, respectively, on Iba-1-ir cell number and area, as determined by Bonferroni posthoc test followed by 2-way ANOVA. α and # indicate a significant difference of acute SF + vehicle from control + vehicle and acute SF + 6-OHDA, respectively, while $ indicates a significant difference between the control mice subjected to vehicle or 6-OHDA treatment, as determined by Bonferroni posthoc test following general linear model analysis of microglia ramification. For statistical significance, alpha (α) was set at 0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure5.compressed.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/4d3be6e586b8aa949f9ee609.png\"},{\"id\":19788287,\"identity\":\"f435d99c-3bfa-437e-be9d-76fbf6fb6278\",\"added_by\":\"auto\",\"created_at\":\"2022-03-30 19:10:30\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":751218,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eChronic Sleep fragmentation and one week of recovery altered microglia activation. \\u003c/em\\u003e\\u003c/strong\\u003eMean (± SE) Iba-1-ir cell number (a, e, i), cell area (b, f, j), and ramification as determined by sholl analysis (c-d, g-h, k-l) in cortex (cingulated cortex; upper panel), hippocampus (dentate gyrus; middle panel) and hypothalamus (pre-optic area; lower panel) of vehicle-injected or chemically sympathectomized mice subjected to control, chronic SF, or a recovery of 1 week following chronic SF (n = 5/group except vehicle treated chronic SF + recovery group of n = 4 mice). The magnified view in each micrograph represents the ramified microglia (magnification: 1000x) subjected to sholl analysis. Asterisk (*) and pound (#) indicate a significant effect of SF and chemical sympathectomy, respectively, on Iba-1-ir cell number and area, as determined by Bonferroni posthoc test followed by 2-way ANOVA. General linear model followed by Bonferroni posthoc test tested the effects on microglia ramification. α and β indicate a significant difference of acute SF from control and recovery mice, respectively, while γ indicates a significant difference between recovery and control mice. Drug-induced effects are indicated by # and \\u0026amp;, presenting a significant difference between vehicle and 6-OHDA treated mice of chronic SF and recovery, respectively. For statistical significance, alpha (α) was set at 0.05.\\u003c/p\\u003e\\u003cp\\u003e\\u003cbr\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure6.compressed.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/b395876618a8140e26e5c282.png\"},{\"id\":21420613,\"identity\":\"1cda3712-2477-4051-9af2-909ea006c690\",\"added_by\":\"auto\",\"created_at\":\"2022-05-13 06:59:35\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":2507343,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-1336087/v2/f6e12b61-05cb-4d06-985e-b754302a4269.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Peripheral sympathectomy alters neuroinflammatory responses and microglial activity in response to sleep fragmentation in female mice\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eThere is accumulating evidence that sleep dysfunction promotes inflammatory responses in brain and peripheral tissues, which can contribute to the development of chronic pathologies, such as metabolic and cardiovascular diseases and neurological disorders [\\u003cspan additionalcitationids=\\\"CR2\\\" citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. The global increase in obesity rates has led to a pronounced rise in the incidence of obstructive sleep apnea [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. Chronic sleep fragmentation (SF) simulates the interrupted sleep experienced by patients with obstructive sleep apnea and other sleep disorders [\\u003cspan additionalcitationids=\\\"CR6\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Concomitantly, sleep loss can increase autonomic sympathetic nervous system (SNS) activation, culminating in the release of norepinephrine (NE) from sympathetic nervous terminals, and NE and epinephrine (E) from the adrenal medullae. These catecholamines are immunomodulatory [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e], and we have recently documented that pharmacological suppression of SNS ameliorates the inflammatory response in peripheral tissues of female mice exposed to acute and chronic sleep dysfunction [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. However, the role that the SNS plays in mediating neuroinflammatory responses to sleep dysfunction is unexplored.\\u003c/p\\u003e \\u003cp\\u003eSleep loss-induced neurological damage and cognitive decline primarily involves microglia activation in the brain [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Microglia cells are resident phagocytes of the brain that act as sentinels of the nervous system to clean debris and regulate inflammatory responses to injury and infection [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Sleep deprivation can promote microglia activation in the absence of injury or infection [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], leading to release of pro-inflammatory cytokines and/or increased phagocytosis [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. Microglia are activated in hippocampus after 5 days of sleep deprivation in rats [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], but acute sleep loss of 8 hours in mice is not sufficient to activate cortical microglia [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. Thus, the temporal and special dynamics of microglia activity during sleep disturbances and deprivation are not fully elucidated.\\u003c/p\\u003e \\u003cp\\u003eIn the present study we examined the effects of acute and chronic SF on neural inflammatory responses in female mice. Females were chosen because of their general underrepresentation in neurobiological studies [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. We also assessed whether 7 days of recovery sleep could sufficiently diminish neuroinflammation from chronic SF. To examine the effect of SNS activation on neuroinflammatory responses to SF, we peripherally sympathectomized mice using 6-hydroxydopamine (6-OHDA). We assessed pro-inflammatory cytokine gene expression and microglial morphology in three distinct brain regions (pre-frontal cortex, hippocampus and hypothalamus) that have been explored in previous studies using experimental sleep fragmentation [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e]. Since peripheral signals of inflammation are proposed to affect neuroinflammation via the neural transmission of vagal afferents reacting to circulating pro-inflammatory agents or the pro-inflammatory agents themselves crossing the blood-brain barrier to directly affect microglia [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], we hypothesized that peripheral sympathectomy would alleviate the neuroinflammatory responses and diminish microglial activity to acute and chronic SF. Finally, we predicted that a 1-week recovery from chronic SF would reverse neuroinflammatory responses as was previously observed when assessing peripheral tissues [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e\"},{\"header\":\"Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnimals and experimental protocol\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFemale C57BL/6J mice between 8-12 weeks of age were used. \\u0026nbsp;Sleep fragmentation (SF) experiments were performed using automated SF chambers (Lafayette Instrument Company; Lafayette, IN; model 80390) with a thin layer of corn bedding as previously described [7]. These chambers ensure that mice are subjected to sleep fragmentation and not absolute sleep deprivation [12]. \\u0026nbsp; Mice were provided \\u003cem\\u003ead libitum\\u003c/em\\u003e access to water and food and housed in a 12:12-h light-dark photoperiod (lights on- 8am, 21\\u0026deg;C \\u0026plusmn; 1\\u0026deg;C) at Western Kentucky University. Subjects were acclimated to the SF chambers for 72 h prior to the commencement of sleep fragmentation experiments to diminish any carryover effects from the novel cage environment[14]. \\u0026nbsp;This study was conducted under the approval of the Institutional Animal Care and Use Committee at Western Kentucky University (#15-11), and procedures followed the National Institutes of Health\\u0026rsquo;s \\u0026ldquo;Guide for the Use and Care of Laboratory Animals\\u0026rdquo; and ARRIVE guidelines.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcute Sleep Fragmentation (Acute SF)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFemale mice (n = 26) were chemically sympathectomized with a daily subcutaneous injection of 6-OHDA (0.1mg/g body mass/day) for 5 days while the remaining mice (\\u003cem\\u003en\\u003c/em\\u003e =26) were injected with the same volume of vehicle (100 \\u0026micro;l of 0.9% NaCl/day). Following the final injection on Day 5, experimental mice (\\u003cem\\u003en\\u003c/em\\u003e = 26; \\u003cem\\u003en\\u003c/em\\u003e = 13 injected with vehicle and \\u003cem\\u003en\\u003c/em\\u003e = 13 injected with 6-OHDA) were subjected to acute SF i.e. a sweeping bar set to move horizontally every 120 seconds for 24 hrs, which simulates the rate of severe sleep apnea among humans ([15]. Controls (\\u003cem\\u003en\\u003c/em\\u003e = 26; \\u003cem\\u003en\\u003c/em\\u003e = 13 injected with vehicle and \\u003cem\\u003en\\u003c/em\\u003e = 13 injected with 6-OHDA) received no sweeping bar movements. 6-OHDA treatment was effective in suppressing SNS activity because serum epinephrine levels were reduced in SF mice receiving 6-OHDA \\u0026nbsp;[7].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eChronic Sleep Fragmentation (Chronic SF)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo induce chronic sleep fragmentation, mice (\\u003cem\\u003en\\u003c/em\\u003e = 28) were subjected to a horizontal sweeping bar set to move every 120 seconds (30 swipes/h) during the light phase (i.e. from 8 am to 8 pm) every day for 8 weeks. To account for increased activity from daily chronic SF, control mice (\\u003cem\\u003en\\u003c/em\\u003e = 26) were subjected to the same number of bar sweeps as experimental mice for only 3 hours of the light phase (i.e. from 8 am to 11am), albeit at 4 times the rate i.e. 2 swipes/minute, to control for daily activity induced by the bar movement. Mice were subcutaneously injected with either 6-OHDA (0.1mg/g body mass/day; \\u003cem\\u003en\\u003c/em\\u003e = 13 of SF mice, \\u003cem\\u003en\\u003c/em\\u003e = 13 of control mice) or vehicle (100 \\u0026micro;l of 0.9% NaCl/day; \\u003cem\\u003en\\u0026nbsp;\\u003c/em\\u003e= 15 of SF mice, \\u003cem\\u003en\\u0026nbsp;\\u003c/em\\u003e= 13 of control mice) between 8-9 am for 5 consecutive days prior to termination of the experiment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eChronic Sleep Fragmentation + Recovery (Chronic SF + R)\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMice were subjected to the chronic SF protocol above, but post SF, control (\\u003cem\\u003en\\u003c/em\\u003e = 16) and SF mice (\\u003cem\\u003en\\u003c/em\\u003e = 25) were subjected to a recovery period (no bar movement) for 7 days during which they were injected with either 6-OHDA (0.1mg/g body mass/day; \\u003cem\\u003en\\u003c/em\\u003e = 13 of chronic SF + R mice, \\u003cem\\u003en\\u003c/em\\u003e = 8 of control mice) or vehicle (100 \\u0026micro;l of 0.9% NaCl/day; \\u003cem\\u003en\\u003c/em\\u003e = 12 of SF + R mice, \\u003cem\\u003en\\u003c/em\\u003e = 8 of control mice) for 5 days.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eFor immunohistochemical studies, the experimental protocols mentioned above were followed, except that the chronic SF+ R experiment did not have a separate control group. Hence, in immunohistochemistry experiment 2, there were three sleep paradigms: controls, chronic SF and chronic SF+ R, each with vehicle or 6-OHDA treatment (\\u003cem\\u003en\\u003c/em\\u003e = 5 per group except vehicle injected chronic SF+ recovery group with \\u003cem\\u003en\\u003c/em\\u003e = 4).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eGene expression analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn all three experiments, 24 hours following the final drug administration, brains of mice (\\u003cem\\u003en\\u003c/em\\u003e = 8/ group except chronic SF + vehicle group of \\u003cem\\u003en\\u003c/em\\u003e = 10 mice) were sampled for RTPCR or immunohistochemistry analysis. Sample sizes of 10 were deemed necessary according to previous power analyses that demonstrate inherent variability in cytokine gene expression. For measurement of brain gene expression, brains were dissected from decapitated mice and stored in RNAlater solution (ThermoFischer Scientific) at 4\\u0026deg;C until RNA extraction.\\u003c/p\\u003e\\n\\u003cp\\u003eRNA was extracted from hippocampus, pre-frontal cortex and hypothalamus using a RNeasy mini kit (Qiagen). RNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (ThermoFischer Scientific). Total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Life Technologies, Cat number: 4368813). The prepared cDNA was used as template for determining relative cytokine gene expression using an ABI 7300 RTPCR system. Cytokine probes (\\u003cem\\u003eIL1\\u003c/em\\u003e\\u003cem\\u003e\\u0026beta;\\u003c/em\\u003e, \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNF\\u0026alpha;\\u003c/em\\u003e, \\u003cem\\u003eTGF\\u0026beta;\\u003c/em\\u003e; Applied Biosystems) labelled with fluorescent marker 5-FAM at the 5\\u0026rsquo; end and quencher MGB at the 3\\u0026rsquo;end were used for genes of interest along with 18S (primer-limited VIC-labelled probe) as the endogenous control according to the manufacturer\\u0026rsquo;s instructions. \\u0026nbsp; Samples were run in duplicate and the fold change in mRNA levels was calculated as the relative mRNA expression levels, 2\\u003csup\\u003e-\\u0026Delta;\\u0026Delta;Ct\\u003c/sup\\u003e [16]. Briefly, the cycle threshold (Ct) obtained by fluorescence exceeding background levels was used to calculate \\u0026Delta;Ct(Ct[target gene] - Ct[18s]). Each Ct value was normalized against the lowest Ct value of a control sample. The negative value of this powered to 2 (2\\u003csup\\u003e-\\u0026Delta;\\u0026Delta;Ct\\u003c/sup\\u003e) was plotted.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eImmunohistochemistry\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor immunohistochemistry studies, 24 hours following final drug administration, mice were deeply anesthetized using isoflurane vapors and then transcardially perfused (\\u003cem\\u003en\\u003c/em\\u003e = 5/group except recovery + vehicle group of \\u003cem\\u003en\\u003c/em\\u003e = 4) with ice-cold saline (0.9%) followed by 4% paraformaldehyde prepared in phosphate buffered saline (PBS). Brains were dissected out from perfused mice, post-fixed in the same fixative overnight, cryoprotected in 30% sucrose and stored at -80\\u0026deg;C until cryosectioned.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e30 \\u0026micro;m sections from brains of control (\\u003cem\\u003en\\u003c/em\\u003e = 5/vehicle or 6-OHDA treatment) and acute SF (\\u003cem\\u003en\\u003c/em\\u003e = 5/vehicle or 6-OHDA treatment) mice from the first study, and control (\\u003cem\\u003en\\u003c/em\\u003e = 5/vehicle or 6-OHDA treatment), chronic SF (\\u003cem\\u003en\\u003c/em\\u003e = 5/vehicle or 6-OHDA treatment) and chronic SF+ R (\\u003cem\\u003en\\u003c/em\\u003e = 4 injected with vehicle, \\u003cem\\u003en\\u003c/em\\u003e = 5 injected with 6-OHDA) mice from the second study were processed for immunohistochemistry of IBA-1. Sections were rinsed thrice in 10mM PBS, followed by an incubation in blocking solution (2.5% goat serum, 0.1% triton X) for 2 hours and overnight incubation (at 4\\u0026deg;C) in the same blocking solution containing anti-Iba-1 (1:300, Catalogue number 019-19741, Wako Pure Chemicals Industries, China). Sections were then incubated with goat anti-rabbit secondary antibody (Alexa fluor 594, catalogue number A11037, Invitrogen, Life Technologies Corporation, Oregon, USA) for 2 hours, followed by washing of sections with 10mM phosphate buffer saline and mounting on slides. After 2 hours of air drying in a dark chamber, slides were mounted and permanently fixed with DAPI+antifade (Vectashield, H-1500, Vector Laboratories, Inc., CA 94010, USA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eImage Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo quantify microglia, every sixth section of the brain was selected and immune-labelled with Iba1 antibody. Iba1 immunoreactivity (-ir) was visualized through a Zeiss Axioplan 2 epifluorescence microscope fitted with a Leica DFC300 FX digital camera. Omission of primary antibodies in control studies confirmed the absence of nonspecific immunoreactivity (data not shown). Iba-1-ir cells in captured images were manually counted by two operators blind to the experimental conditions using the cell counting plug-in in Image J (Version 1.45 J; National Institutes of Health, Bethesda, MD, USA). The brightness and contrast of images were adjusted to a firm setting for all sections to aid in visualization of immunostaining. A box of size 80000 \\u0026micro;m\\u003csup\\u003e2\\u003c/sup\\u003e, 100000 \\u0026micro;m\\u003csup\\u003e2\\u003c/sup\\u003e and 42000 \\u0026micro;m\\u003csup\\u003e2\\u003c/sup\\u003e was overlaid on 100x (10x objective, 10x eyepiece) pictures of sections with cingulated cortex (Bregma: +0.74: +0.38), dentate gyrus (Bregma: -1.64: -2.12) and pre-optic area (Bregma: -0.94: -1.28), respectively, and region identification was based on the mouse brain atlas (Paxinos and Franklin, 2001; http://www.mbl.org/atlas170/atlas170_frame.html). Cells were counted in a total of 4 sections. Iba1-ir values were averaged from both sides of bilateral nuclei, and the count from 4 sections was summed for each mice. Total numbers of positive cells per animal were multiplied by six to estimate the number of cells per cingulate cortex, dentate gyrus and pre-optic area.\\u003c/p\\u003e\\n\\u003cp\\u003eFor morphological analysis of microglia, all analysis were performed on maximum intensity projections (Z-project, maximum intensity function in Image J) of the Z-stacks collected with 0.7\\u0026micro;m increments on a Olympus Flowview (FV1000) confocal microscope with 10X eyepiece and 100X objective (1000X). Microglia were classified as ramified (numerous thin processes, radial branching), primed (thickened processes, increased polarity, and proliferation with reduced secondary branching), reactive (thickened stout processes with highly reduced branching), or amoeboid (rounded soma with no branching; Figure 1A\\u0026ndash;D) based on standard morphological criteria [17]. Sholl analysis was performed over 20 concentric circles, with the inner radius of 6\\u0026micro;m and outer radius of 35\\u0026micro;m on a total of 15 ramified microglia (5 from cingulated cortex + 5 from dentate gyrus + 5 from pre-optic area) per brain and 5 brain/group. Analysis was done by 3 operators blind to the experimental conditions. Non-overlapping microglia cells that were in a ramified state exhibiting intact microglial processes unobscured by either background labelling or other cells were photographed and analysed.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eData are presented as mean (\\u0026plusmn;SE). All statistical analyses were done using GraphPad prism (version 6.0), IBM SPSS Statistics version 20 software as appropriate. Two-way ANOVAs assessed the effect of sleep fragmentation (factor 1), 6-OHDA treatment (factor 2) and their interaction on mRNA expression of cytokines and Iba-1 immunohistochemistry in brain. A three-factor analysis, using Univariate General linear model (GLM) tested the effects of sleep fragmentation (factor 1), 6-OHDA treatment (factor 2), and distance from soma (factor 3), and their interactions on morphology of the Iba-1 staining. Bonferroni multiple comparisons were used for posthoc analysis. \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e\\u0026lt; 0.05 was considered statistically significant.\\u0026nbsp;\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eSF-induced effects on cytokine mRNA expression levels in brain\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThere was a significant effect of acute SF on \\u003cem\\u003eIL-1β\\u003c/em\\u003e and \\u003cem\\u003eIL-6\\u003c/em\\u003e expression levels in prefrontal cortex (\\u003cem\\u003eIL-1β\\u003c/em\\u003e: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 4.69, \\u003cem\\u003ep\\u003c/em\\u003e = 0.039, \\u003cem\\u003eIL-6\\u003c/em\\u003e: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 8.07, \\u003cem\\u003ep\\u003c/em\\u003e = 0.008, 2-way ANOVA), while acute SF induced effects on \\u0026nbsp;\\u003cem\\u003eIL-1β\\u0026nbsp;\\u003c/em\\u003eexpression levels in both prefrontal cortex and hippocampus were dependent on 6-OHDA treatment (prefrontal cortex: interaction- \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 7.81, \\u003cem\\u003ep\\u003c/em\\u003e = 0.009, hippocampus: interaction - \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,26\\u003c/sub\\u003e = 6.52, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0169, 2-way ANOVA). Similarly, in hypothalamus, there was a significant effect of acute SF on \\u003cem\\u003eIL-1β\\u003c/em\\u003e (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 6.07, \\u003cem\\u003ep\\u003c/em\\u003e = 0.020), and an interaction of acute SF and 6-OHDA on \\u003cem\\u003eIL-6\\u0026nbsp;\\u003c/em\\u003e(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 16.21, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0004; 2-way ANOVA). \\u003cem\\u003eTNFα\\u003c/em\\u003e levels were unaffected by the experimental treatment in brain tissues while \\u003cem\\u003eTGFβ\\u003c/em\\u003e levels in all three brain regions were significantly affected by acute SF (prefrontal cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 8.62, \\u003cem\\u003ep\\u003c/em\\u003e = 0.007; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 14.29,\\u003cem\\u003e\\u0026nbsp;p\\u003c/em\\u003e = 0.0008; hypothalamus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 15.07, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0006) and 6-OHDA treatment (except in prefrontal cortex; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 14.77, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0006, hypothalamus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 10.55, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0030; 2-way ANOVA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThere was a significant effect of chronic SF on the expression levels of all genes assessed in prefrontal cortex (except \\u003cem\\u003eTNFα\\u003c/em\\u003e; \\u003cem\\u003eIL-1β\\u003c/em\\u003e: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,30\\u003c/sub\\u003e = 4.29, \\u003cem\\u003ep\\u003c/em\\u003e = 0.047; \\u003cem\\u003eIL-6\\u003c/em\\u003e: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,29\\u003c/sub\\u003e = 7.512, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0106; \\u003cem\\u003eTGFβ\\u003c/em\\u003e: \\u0026nbsp; \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,29\\u003c/sub\\u003e = 6.510, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0163) along with a significant effect of 6-OHDA treatment and its interaction with chronic SF on \\u003cem\\u003eTNFα\\u003c/em\\u003e and \\u003cem\\u003eIL-1β\\u003c/em\\u003e, respectively (\\u003cem\\u003eTNFα\\u003c/em\\u003e: 6-OHDA- \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,31\\u003c/sub\\u003e = 4.94, \\u003cem\\u003ep\\u003c/em\\u003e = 0.034; \\u003cem\\u003eIL-1β\\u003c/em\\u003e: interaction- \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,30\\u003c/sub\\u003e = 4.498, \\u003cem\\u003ep\\u003c/em\\u003e = 0.042). Further, hippocampal \\u003cem\\u003eTGFβ\\u0026nbsp;\\u003c/em\\u003eexpression was significantly affected by 6-OHDA (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,30\\u003c/sub\\u003e = 4.43, \\u003cem\\u003ep\\u003c/em\\u003e = 0.044) and its interaction with chronic SF (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,30\\u003c/sub\\u003e = 6.17, \\u003cem\\u003ep\\u003c/em\\u003e = 0.019; 2-way ANOVA). Similarly, hypothalamic \\u003cem\\u003eIL-1β\\u003c/em\\u003e expression was significantly affected by 6-OHDA treatment (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,29\\u003c/sub\\u003e = 19.11, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0002), and hypothalamic \\u003cem\\u003eTNFα\\u0026nbsp;\\u003c/em\\u003elevels were significantly affected by chronic SF (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,29\\u003c/sub\\u003e = 8.27, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0076) and 6-OHDA (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,29\\u003c/sub\\u003e = 28.16, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; 2-way ANOVA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eOne week of recovery sleep significantly affected \\u003cem\\u003eIL-1β\\u003c/em\\u003e expression in hippocampus (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 5.97, \\u003cem\\u003ep\\u003c/em\\u003e = 0.021) and hypothalamus (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 5.13, \\u003cem\\u003ep\\u003c/em\\u003e = 0.032), along with a significant effect of recovery and 6-OHDA treatment on \\u003cem\\u003eTNFα\\u003c/em\\u003e expression in brain (hippocampus: recovery - \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 51.91, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001, 6-OHDA- \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 6.49, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0166; hypothalamus: recovery - \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 4.36, \\u003cem\\u003ep\\u003c/em\\u003e = 0.046, 6-OHDA- \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 4.84, \\u003cem\\u003ep\\u003c/em\\u003e = 0.036). \\u003cem\\u003eIL-6\\u003c/em\\u003e expression levels in prefrontal cortex and hippocampus were significantly affected by recovery (prefrontal cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 14.38, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0007; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 7.97, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0087) and 6-OHDA treatment (prefrontal cortex: F\\u003csub\\u003e1,28\\u003c/sub\\u003e = 14.98, p = 0.0006; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 23.54, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001), the expression levels in hypothalamus were significantly affected by 6-OHDA (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 42.39, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001) and its interaction with recovery (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 10.30, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0034). Neural \\u003cem\\u003eTGFβ\\u0026nbsp;\\u003c/em\\u003eexpression levels were significantly affected by recovery (hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 11.00, \\u003cem\\u003ep\\u003c/em\\u003e = 0.003; hypothalamus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 11.37, \\u003cem\\u003ep\\u003c/em\\u003e = 0.002), 6-OHDA (prefrontal cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 5.56, \\u003cem\\u003ep\\u003c/em\\u003e = 0.026; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 12.75, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001; hypothalamus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 14.48, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0007) and their interaction (prefrontal cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 12.68, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0013; hypothalamus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,28\\u003c/sub\\u003e = 13.81, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0009), except the effect of recovery and its interaction with 6-OHDA in prefrontal cortex and hippocampus, respectively (2-way ANOVA).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn comparison to controls, \\u003cem\\u003eIL-1β\\u003c/em\\u003e expression under both SF regimes was significantly increased in brain regions (except hippocampus of chronic SF exp; Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 2a, e, i, 3a, i). Acute SF caused a significant reduction in expression levels of \\u003cem\\u003eTGFβ\\u003c/em\\u003e in all three brain tissues, and in \\u003cem\\u003eIL-6\\u003c/em\\u003e in prefrontal cortex and hypothalamus (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 2b, d, f, h, j, l), as opposed to chronic\\u0026nbsp;SF which lead to a significant increase in \\u003cem\\u003eIL-6\\u0026nbsp;\\u003c/em\\u003eexpression in prefrontal cortex, \\u003cem\\u003eTNFα\\u0026nbsp;\\u003c/em\\u003ein hypothalamus and\\u0026nbsp;\\u003cem\\u003eTGFβ\\u003c/em\\u003e in hippocampus (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 3b, h, k).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eA recovery period of 1 week after chronic\\u0026nbsp;SF was enough to negate the effects of chronic\\u0026nbsp;SF (except \\u003cem\\u003eIL-6\\u003c/em\\u003e in prefrontal cortex). In fact, \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNFα\\u003c/em\\u003e and \\u003cem\\u003eTGFβ\\u003c/em\\u003e expression in hippocampus and hypothalamus and \\u003cem\\u003eIL-1β\\u003c/em\\u003e expression in hypothalamus was significantly lower in recovery mice compared with controls (Bonferroni posthoc comparison, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 4f-l). In addition, lack of significant differences between 6-OHDA administered controls and experimental groups in all experiments (except hypothalamic \\u003cem\\u003eTNFα\\u003c/em\\u003e of chronic\\u0026nbsp;SF+R experiment), along with significant reduction in hypothalamic \\u003cem\\u003eIL-1β\\u003c/em\\u003e and \\u003cem\\u003eTNFα\\u0026nbsp;\\u003c/em\\u003ein 6-OHDA-treated chronic SF mice, and in \\u003cem\\u003eTGFβ\\u003c/em\\u003e in hippocampus and cortex of 6-OHDA treated acute and chronic SF mice, indicated that chemical sympathectomy significantly attenuated SF-induced neuroinflammatory responses (Bonferroni posthoc test, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e\\u0026lt; 0.05, fig. 2-4).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSF-induced effects on microglia activity\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIba-ir cell number was significantly affected by acute\\u0026nbsp;SF (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 15.60, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 14.79, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 6.03, \\u003cem\\u003ep\\u003c/em\\u003e = 0.026), 6-OHDA treatment (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 15.60, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 16.33, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001), and their interaction (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 26.49, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 10.45, \\u003cem\\u003ep\\u003c/em\\u003e = 0.005; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,16\\u003c/sub\\u003e = 5.56, \\u003cem\\u003ep\\u003c/em\\u003e = 0.031) in\\u0026nbsp;all three brain regions, except an effect of 6-OHDA in pre-optic area, as determined by 2-way ANOVA. In all three brain regions, acute SF significantly increased Iba-ir, which was significantly decreased with 6-OHDA treatment (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 5a, e, i). Similarly, Iba-ir soma area was significantly affected by acute SF in pre-optic area\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,96\\u003c/sub\\u003e = 6.46, \\u003cem\\u003ep\\u003c/em\\u003e = 0.012), by 6-OHDA treatment in cortex\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,96\\u003c/sub\\u003e = 12.04, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0008), and by their interaction in hippocampus\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,96\\u003c/sub\\u003e = 4.87, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e= 0.029)\\u0026nbsp;and pre-optic area (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,96\\u003c/sub\\u003e = 4.07, \\u003cem\\u003ep\\u003c/em\\u003e = 0.047;\\u0026nbsp;2-way ANOVA), with significantly greater –ir soma area in pre-optic area of mice subjected to acute SF compared with controls (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 5b, f, j). Iba-ir soma area in cortex of acute SF mice was significantly greater than their counterparts pre-treated with 6-OHDA (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 5b).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn the second experiment, there was a significant effect of SF on Iba-ir cell number in cortex\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,23\\u003c/sub\\u003e = 26.24, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e\\u0026lt; 0.0001)\\u0026nbsp;and hippocampus\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,23\\u003c/sub\\u003e = 4.43, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e= 0.023), of 6-OHDA treatment and its interaction with SF in cortex (6-OHDA: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,23\\u003c/sub\\u003e = 8.10,\\u003cem\\u003e\\u0026nbsp;p\\u003c/em\\u003e = 0.009; SF x 6-OHDA: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,23\\u003c/sub\\u003e = 19.30, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; 2-way ANOVA). Interestingly, Iba-ir cell number in cortex and hippocampus of mice subjected to chronic SF \\u0026gt; recovery \\u0026gt; controls (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05 Fig. 6a, e). Furthermore, 6-OHDA treatment significantly reduced Iba-ir in mice subjected to chronic SF (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05; Fig. 6a, e, i).\\u0026nbsp;There was a significant effect of SF on Iba-ir soma area in cortex (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,139\\u003c/sub\\u003e = 6.49, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e= 0.002) and hippocampus (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,139\\u003c/sub\\u003e = 4.68, \\u003cem\\u003ep\\u003c/em\\u003e = 0.011), and of 6-OHDA treatment (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,139\\u003c/sub\\u003e = 12.51, \\u003cem\\u003ep\\u003c/em\\u003e = 0.0006) and its interaction with SF (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,139\\u003c/sub\\u003e = 6.73, \\u003cem\\u003ep\\u003c/em\\u003e = 0.002) on Iba-ir soma area in pre-optic area (2-way ANOVA). Iba-ir soma area was significantly increased in chronic SF mice compared with recovery and controls (except in hippocampus; Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 6b, f, j). Further, chronic SF mice had significantly lower Iba-ir soma area in pre-optic area when treated with 6-OHDA (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig. 6j).\\u003c/p\\u003e\\n\\u003cp\\u003eThree-factor univariate general linear models indicated a significant effect of acute SF and 6-OHDA treatment on microglial ramification in\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,1824\\u003c/sub\\u003e = 19.66, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001) and pre-optic area (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,1824\\u003c/sub\\u003e = 43.31, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001). Microglia ramification significantly varied with distance from soma\\u0026nbsp;(cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,1824\\u003c/sub\\u003e = 38.38, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,1824\\u003c/sub\\u003e = 20.97, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,1824\\u003c/sub\\u003e = 10.02, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001)\\u0026nbsp;and acute SF induced effects were dependent on 6-OHDA treatment in all three regions\\u0026nbsp;(acute SF x 6-OHDA interaction - cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,1824\\u003c/sub\\u003e = 6.124, \\u003cem\\u003ep\\u003c/em\\u003e = 0.013; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,1824\\u003c/sub\\u003e = 25.76, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,1824\\u003c/sub\\u003e = 15.99, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001). There was a significant three-way interaction only in pre-optic area (acute SF x 6-OHDA x distance from soma: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,1824\\u003c/sub\\u003e = 3.38, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001, univariate GLM). In cortex, acute SF significantly increased the ramification at 6.5 µm from the centre of soma when compared with controls (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 5c). 6-OHDA significantly affected the microglia ramification in controls, with higher ramification in cortex (between 9 to 16 µm from the soma centre), hippocampus (23 µm from the soma centre) and pre-optic area (at 26 and 29 µm from soma centre; Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 5c, d, g, h, k, l). Similarly, acute SF mice when injected with 6-OHDA, showed higher ramification in hippocampus at a distance of 29-30.5 µm from the centre of soma (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 5g, h).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eChronic SF had more profound effects on microglia morphology with a significant effect of\\u0026nbsp;(cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 108.79, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 24.59, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 72.93, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001), 6-OHDA treatment (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,2754\\u003c/sub\\u003e = 5.14, \\u003cem\\u003ep\\u003c/em\\u003e = 0.023; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,2754\\u003c/sub\\u003e = 16.51, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e1,2754\\u003c/sub\\u003e = 113.17, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001), distance from soma (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,2754\\u003c/sub\\u003e = 40.82, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,2754\\u003c/sub\\u003e = 37.55, \\u003cem\\u003ep\\u0026nbsp;\\u003c/em\\u003e\\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,2754\\u003c/sub\\u003e = 29.74, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001), and SF x 6-OHDA interaction (cortex: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 52.79, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; hippocampus: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 37.78, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001; pre-optic area: \\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e2,2754\\u003c/sub\\u003e = 28.68, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001)\\u0026nbsp;in all three brain regions, as determined by univariate GLM. In addition, there was a significant interaction effect of distance from soma with SF\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e36,2754\\u003c/sub\\u003e = 1.83, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001)\\u0026nbsp;and 6-OHDA treatment\\u0026nbsp;(\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e18,2754\\u003c/sub\\u003e = 2.65, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001)\\u0026nbsp;in cortex and pre-optic area, respectively. There was a significant three-way interaction in hippocampus (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e36,2754\\u003c/sub\\u003e = 1.91, \\u003cem\\u003ep\\u003c/em\\u003e = 0.001) and pre-optic area (\\u003cem\\u003eF\\u003c/em\\u003e\\u003csub\\u003e36,2754\\u003c/sub\\u003e = 2.16, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.0001), but not in cortex. In comparison with controls, chronic SF caused microglia hyper-ramification in all three brain regions (distance from centre of soma: 14 to 32 µm in cortex, 17 to 26 µm in hippocampus, and 20 to 35 µm in pre-optic area). A significantly higher ramification in cortex at 11 to 32 µm, in hippocampus at 26 to 27.5 and 32 to 33.5 µm, and in pre-optic area at 20 to 35 µm from centre of soma was observed in chronic SF mice compared with recovery mice (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 6c, d, g, h, k, l).\\u003c/p\\u003e\\n\\u003cp\\u003eHippocampal microglial response was still observed after 1 week of recovery, as ramification was significantly higher in mice subjected to recovery at 12.5 to 14 µm distance from centre of soma (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 6g, h). Further, 6-OHDA administration diminished the neuroinflammatory response from chronic SF, as evidenced by significantly lower microglia ramification in cortex (at 23 to 33.5 µm distance from centre of soma), hippocampus (at 17 to 23 µm, 26 and 30.5 µm distance from centre of soma) and pre-optic area (at 14, and 20 to 30.5 µm distance from centre of soma) in mice subjected to 6-OHDA treatment following chronic SF, relative to their counterparts injected with vehicle (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 6c, d, g, h, k, l). The pre-optic area of recovery mice injected with 6-OHDA had lower microglia ramification at 11 to 14 µm distance from centre of soma, compared with recovery mice not injected with 6-OHDA (Bonferroni posthoc test, \\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, Fig 6k, l).\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eSleep disturbances can promote neuroinflammation and blood-brain barrier disruption mediated by inflammatory molecules produced from astrocytes and microglia. For instance, sleep restriction for 72 h elevated \\u003cem\\u003eIL-1β\\u003c/em\\u003e, \\u003cem\\u003eIL-6\\u003c/em\\u003e and \\u003cem\\u003eTNFα\\u003c/em\\u003e in hippocampus and basal forebrain of rats [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e] and chronic sleep restriction for 21 days elevated \\u003cem\\u003eIL-6\\u003c/em\\u003e and \\u003cem\\u003eTNFα\\u003c/em\\u003e in hippocampus and \\u003cem\\u003eTNFα\\u003c/em\\u003e in prefrontal cortex of rats [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. Similarly, in the present study, sleep fragmentation (SF) promoted neuroinflammatory responses albeit in a brain region-specific and SF-duration dependent manner. In addition to having proinflammatory roles, elevated \\u003cem\\u003eIL-1β\\u003c/em\\u003e in brain following acute and chronic SF (except in hippocampus), and \\u003cem\\u003eTNFα\\u003c/em\\u003e in hypothalamus following chronic SF may indicate an accumulation of SRS (sleep regulatory substances) following a period of prolonged wakefulness [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e]. The increase in neural \\u003cem\\u003eTNFα\\u003c/em\\u003e response after chronic but not acute SF is in corroboration with previous reports [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. An induction of an anti-inflammatory environment in the brain after long-term SF has been reported before [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. Increased hippocampal \\u003cem\\u003eTGFβ\\u003c/em\\u003e expression following chronic SF perhaps provided evidence for neuroprotection from inflammation. Further, while primarily pro-inflammatory, IL-6 has been described to play anti-inflammatory roles in some scenarios, causing stimulation of IL-10 synthesis [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] and inhibition of IL-1 and TNFα production [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. While the precise functioning of IL-6 is not deciphered in the present study, opposite trends in its mRNA expression under acute and chronic SF seems to suggest an effort to closely regulate neuroinflammatory response to SF.\\u003c/p\\u003e \\u003cp\\u003eMicroglial activation is considered a main source of neuroinflammation among other factors [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. Sleep loss has been shown to affect microglial morphology[\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. In mature CNS, microglia are in a \\u0026lsquo;resting state\\u0026rsquo; with highly ramified and motile processes, continuously surveying the surrounding environment. In response to injury, microglia shift from resting to a primed phagocytic \\u0026lsquo;activated\\u0026rsquo; state [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Sleep loss, in the absence of injury, has been reported to increase phagocytic activity of microglia [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. On the other hand, psychological and mechanical stress has also been shown to increase the microglial secondary branching in the cortex of rats [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e], thus suggesting that microglial activation in response to homeostatic perturbations can range from mild non-inflammatory hyper-ramified phenotype to pro-inflammatory, apoptotic and phagocytic phenotypes [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. Unlike chronic OSA and SF, neither OSA nor sleep deprivation/restriction of 1 night has been previously reported to increase the phagocytic activity of microglia [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. Therefore, we surmise that perhaps acute SF of 1 night might affect the mild \\u0026lsquo;non-inflammatory\\u0026rsquo; instead of the \\u0026lsquo;activated\\u0026rsquo; phagocytic morphology of microglia. Absence of microglial hyper-ramification in acute SF mice suggested that SF-induced neuroinflammatory changes may not necessarily be associated with changes in microglial morphology [\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. On the other hand, increased ramification after chronic SF suggests that chronic sleep perturbations induced microglial morphological changes that may include an increase in \\u0026lsquo;activated\\u0026rsquo; phenotype involved in enhanced phagocytosis of synaptic elements [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e] and an increased ramification of the \\u0026lsquo;resting\\u0026rsquo; phenotype possibly involved in increased surveillance and scanning of microenvironment [\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. Given the reversibility and complexity of microglia morphology, especially across brain regions [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], we analysed only the ramified morphology of microglia. SF-induced alterations in the phagocytic phenotype need to be tested in future studies, and thus presently cannot be ruled out.\\u003c/p\\u003e \\u003cp\\u003eUsually, inflammation from sleep loss return to basal levels after sleep recovery. However, depending on duration of sleep loss and recovery, some immune components may remain altered after sleep recovery [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. In this study, while seven days of recovery completely alleviates inflammatory responses in peripheral tissues from a previous study [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], it was not sufficient to restore homeostasis in brain. Elevated Iba-ir in all three brain regions and increased \\u003cem\\u003eIL-6\\u003c/em\\u003e expression in cortex of recovery mice indicated that neuroinflammatory responses persisted at least 1 week following chronic SF. Consistent with our finding, a recent study reported that even after three weeks of recovery from sleep loss, neuronal apoptosis, microglial activation and IL-6 response still occurred in hippocampus of mice [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. Furthermore, similar to our results, anti-inflammatory responses lower than baseline levels have been reported in mice subjected to 20 days of recovery post SD of 192 hours [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. In addition, we documented a significant reduction in cytokine gene expression in brain after 7 days of recovery sleep. Perhaps, these changes may be a compensatory response that limits neurological and cardiovascular dysfunction related to SF-induced hyperactivation of inflammation over prolonged periods of SF. Irrespective of direction of deviation from baseline, it is plausible that one possible mediator of delayed return of neuroinflammatory to baseline homeostatic state was SNS and not the hypothalamic-pituitary-adrenal axis, since high levels of NE were found after 1 week of recovery sleep [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eOur initial hypothesis that the SNS contributes to neuroinflammatory responses to sleep loss has been supported, as chemical sympathectomy significantly altered the inflammatory responses in all three SF paradigms examined. These results reiterate the complexities of potential NEI (neuro-endocrine-immune) interactions by which SF can affect immune functioning [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Peripheral sympathectomy also successfully alleviated SF-induced neuroinflammatory responses. Perhaps one or more of the following established mechanisms contributed to these results: (1) a passive diffusion of peripheral cytokines to brain via circumventricular organs or an active transport with carrier proteins across blood brain barrier (BBB) affected the microglial activity and neuroinflammatory processes (2) peripheral signals stimulated the endothelial cells of BBB to secrete molecules affecting activity of neurons and glia and/or (3) the autonomic nervous system itself via vagal afferents activated the neuroinflammatory responses [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eInterestingly, 6-OHDA treatment also altered baseline cytokine levels. More specifically, \\u003cem\\u003eTGFβ\\u003c/em\\u003e in hippocampus and \\u003cem\\u003eIL-6\\u003c/em\\u003e in hypothalamus was significantly lowered, while \\u003cem\\u003eIL-1β\\u003c/em\\u003e in hippocampus was significantly increased in control mice after chemical sympathectomy in acute SF experiment. \\u003cem\\u003eIL-6\\u003c/em\\u003e, \\u003cem\\u003eTNFα\\u003c/em\\u003e and \\u003cem\\u003eTGFβ\\u003c/em\\u003e in hypothalamus and hippocampus (except \\u003cem\\u003eTNFα\\u003c/em\\u003e) of control mice were significantly attenuated after 6-OHDA treatment in the recovery experiment. The effects of chemical sympathectomy on baseline immune function in chronic SF experiment were less profound with only a significant reduction in hypothalamic \\u003cem\\u003eTNFα\\u003c/em\\u003e expression in controls treated with 6-OHDA. Such differences between the three experiments could be attributed to the differences in experimental paradigms and duration of treatments involved (cf. Figure\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Lastly, our data suggests that baseline neuroinflammatory responses of hypothalamus and hippocampus were more susceptible to peripheral 6-OHDA treatment than the cortex. Further studies are warranted to understand how peripheral signals activate neuroinflammation across different brain regions.\\u003c/p\\u003e \\u003cp\\u003eHistorically, the bias towards use of male rodents in research has resulted in data gaps to effectively discern the effects of sleep abnormalities upon female physiology. Given the sexual differences in susceptibility to diseases including neurological and immune diseases [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e], this study provides critical insight regarding effects of sleep disruption on female peripheral and neural immune responses. Sex steroids regulate the development and maturation of immune cells and responses [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. As result, inflammatory profiles [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e] and microglial activity [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e] differ between the sexes. Therefore, caution is warranted before extrapolating these results to responses in males. Paradoxical SD affects estrous cyclicity in rats [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Since we the reproductive status was not tested in mice of our study, the involvement of reproductive hormones in regulating SF-induced inflammation and microglial responses cannot be ruled out. Restraint stress also affects phasing of the estrous cycle in female rats, although there is large individual variation [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Because sample sizes in our study were small, future studies should evalute the effect of estrous cycling upon SF-induced inflammation using a greater sample size.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study systematically evaluated the SF-associated neuroinflammation and microglial responses of female mice. Changes in inflammatory responses seemed to be reflective of stress-axes activation and were related to the duration of SF [\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Unlike previous reports, our experimental approach of exposing mice to 8 weeks of SF can more accurately be expected to mimic sleep abnormalities, such as OSA and the diseases associated with it, in the modern world. Moreover, it is important to emphasize that a recovery sleep of seven nights following such chronic sleep fragmentation was clearly insufficient for a full return to homeostasis. Importantly, to our knowledge this is the first study showing chronic SF-induced subtle changes in the morphology of microglia primarily characterised as \\u0026lsquo;resting\\u0026rsquo;. Finally, this study provides evidence of a critical contribution of SNS in development of an inflammatory state. These findings could lead to novel therapeutic interventions that target the SNS for treating inflammation-dependent disorders, such as cardiovascular diseases.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe thank Melanie Richter and Nicholas Wheeler for sampling, and Naomi Rowland for assistance with RTPCR. This research was supported by the National Institutes of Health (R15GM117534) and\\u0026nbsp;the Kentucky Biomedical Research Infrastructure Network (NIGMS grant #8P20GM103436-14) to NTA.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIM wrote the primary draft, performed the experiments and primary analysis, KBP, KE and AS analysed the data, and NTA designed the study, wrote the final draft and acquired funding. All authors read and approved the final manuscript.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe gene expression and image analysis datasets generated for this study are available from Figshare (https://doi.org/10.6084/m9.figshare.19369592.v1). Raw image files are available from the corresponding author on reasonable request due to large file sizes.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eRajaratnam, S. M. \\u0026amp; Arendt, J. Health in a 24-h society. Lancet \\u003cb\\u003e358\\u003c/b\\u003e, 999\\u0026ndash;1005 (2001).\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eSchwartz S., \\u003cem\\u003eet al\\u003c/em\\u003e. 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Immun. \\u003cb\\u003e52\\u003c/b\\u003e, 88\\u0026ndash;97 (2016).\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"6-hydroxydopamine, cytokines, microglia, neuroinflammation, obstructive sleep apnea, sleep fragmentation, sympathetic nervous system\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-1336087/v2\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-1336087/v2\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eSleep loss, either induced by obstructive sleep apnea or other forms of sleep dysfunction, induces an inflammatory response, as commonly measured by increased circulating levels of pro-inflammatory cytokines. Increased catecholamines from sympathetic nervous system (SNS) activation regulates this peripheral inflammation. However, the role that catecholamines play in mediating neuroinflammation from sleep perturbations is undescribed. The aims of this study were to determine (i) the effect of peripheral SNS inhibition upon neuroinflammatory responses to sleep fragmentation (SF) and (ii) whether homeostasis can be restored after 1 week of recovery sleep. We measured gene expression levels of pro- and anti-inflammatory cytokines and microglial activity in brain (prefrontal cortex, hippocampus and hypothalamus) of female mice that were subjected to acute SF for 24 hours, chronic SF for 8 weeks, or 7 days of recovery after chronic SF. In each experiment, SF and control mice were peripherally sympathectomized with 6-OHDA (6-hydroxydopamine) or injected with vehicle. SF elevated cytokine mRNA expression in brain and increased microglial density and cell area in some regions. In addition, chronic SF promoted hyper-ramification in resting microglia upon exposure to chronic, but not acute, SF. Effects of chronic SF were more pronounced than acute SF, and 1 week of recovery was not sufficient to alleviate neuroinflammation. Importantly, 6-OHDA treatment significantly alleviated SF-induced inflammation and microglial responses. This study provides evidence of SNS regulation of neural inflammation from SF, suggesting a potential role for therapeutics that could mitigate neuroinflammatory responses to sleep dysfunction.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Peripheral sympathectomy alters neuroinflammatory responses and microglial activity in response to sleep fragmentation in female mice\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":2,\"date\":\"2022-03-30 19:10:28\",\"doi\":\"10.21203/rs.3.rs-1336087/v2\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}},{\"code\":1,\"date\":\"2022-02-09 20:51:45\",\"doi\":\"10.21203/rs.3.rs-1336087/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"0df49f3b-9771-4a52-bde5-0b7c93ba1f28\",\"owner\":[],\"postedDate\":\"March 30th, 2022\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2022-05-13T06:59:26+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2022-03-30 19:10:28\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v2\",\"identity\":\"rs-1336087\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-1336087\",\"identity\":\"rs-1336087\",\"version\":[\"v2\"]},\"buildId\":\"WrCJVZZCHTDjtuVLN7oU0\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}