Hypocretin Mediates Sleep and Wake Disturbances in a Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of disability worldwide. Post-TBI sleep and wake disturbances are extremely common and difficult for patients to manage. Sleep and wake disturbances contribute to poor functional and emotional outcomes from TBI, yet effective therapies remain elusive. A more comprehensive understanding of mechanisms underlying post-TBI sleep and wake disturbance will facilitate development of effective pharmacotherapies. Previous research in human patients and animal models indicates that altered hypocretinergic function may be a major contributor to sleep-wake disturbance after TBI. In this study, we further elucidate the role of hypocretin by determining the impact of TBI on sleep-wake behavior of hypocretin knockout (HCRT KO) mice. Adult male C57BL/6J and HCRT KO mice were implanted with electroencephalography recording electrodes, and pre-injury baseline recordings were obtained. Mice were then subjected to either moderate TBI or sham surgery. Additional recordings were obtained and sleep-wake behavior determined at 3, 7, 15, and 30 days after TBI or sham procedures. At baseline, HCRT KO mice had a significantly different sleep-wake phenotype than control C57BL/6J mice. Post-TBI sleep-wake behavior was altered in a genotype-dependent manner: sleep of HCRT KO mice was not altered by TBI, whereas C57BL/6J mice had more non-rapid eye movement sleep, less wakefulness, and more short wake bouts and fewer long wake bouts. Numbers of hypocretin-positive cells were reduced in C57BL/6J mice by TBI. Collectively, these data indicate that the hypocretinergic system is involved in the alterations in sleep-wake behavior that develop after TBI in this model, and suggest potential therapeutic interventions.

Hypocretinergic and cholinergic contributions to sleep-wake disturbances in a mouse model of traumatic brain injury.

Disorders of sleep and wakefulness occur in the majority of individuals who have experienced traumatic brain injury (TBI), with increased sleep need and excessive daytime sleepiness often reported. Behavioral and pharmacological therapies have limited efficacy, in part, because the etiology of post-TBI sleep disturbances is not well understood. Severity of injuries resulting from head trauma in humans is highly variable, and as a consequence so are their sequelae. Here, we use a controlled laboratory model to investigate the effects of TBI on sleep-wake behavior and on candidate neurotransmitter systems as potential mediators. We focus on hypocretin and melanin-concentrating hormone (MCH), hypothalamic neuropeptides important for regulating sleep and wakefulness, and two potential downstream effectors of hypocretin actions, histamine and acetylcholine. Adult male C57BL/6 mice (n=6-10/group) were implanted with EEG recording electrodes and baseline recordings were obtained. After baseline recordings, controlled cortical impact was used to induce mild or moderate TBI. EEG recordings were obtained from the same animals at 7 and 15 days post-surgery. Separate groups of animals (n=6-8/group) were used to determine effects of TBI on the numbers of hypocretin and MCH-producing neurons in the hypothalamus, histaminergic neurons in the tuberomammillary nucleus, and cholinergic neurons in the basal forebrain. At 15 days post-TBI, wakefulness was decreased and NREM sleep was increased during the dark period in moderately injured animals. There were no differences between groups in REM sleep time, nor were there differences between groups in sleep during the light period. TBI effects on hypocretin and cholinergic neurons were such that more severe injury resulted in fewer cells. Numbers of MCH neurons and histaminergic neurons were not altered under the conditions of this study. Thus, we conclude that moderate TBI in mice reduces wakefulness and increases NREM sleep during the dark period, effects that may be mediated by hypocretin-producing neurons and/or downstream cholinergic effectors in the basal forebrain.

Sleep fragmentation and sepsis differentially impact blood-brain barrier integrity and transport of tumor necrosis factor-α in aging.

The factors by which aging predisposes to critical illness are varied, complex, and not well understood. Sepsis is considered a quintessential disease of old age because the incidence and mortality of severe sepsis increases in old and the oldest old individuals. Aging is associated with dramatic changes in sleep quality and quantity and sleep increasingly becomes fragmented with age. In healthy adults, sleep disruption induces inflammation. Multiple aspects of aging and of sleep dysregulation interact via neuroimmune mechanisms. Tumor necrosis factor-α (TNF), a cytokine involved in sleep regulation and neuroimmune processes, exerts some of its effects on the CNS by crossing the blood-brain barrier (BBB). In this study we examined the impact of sepsis, sleep fragmentation, and aging on BBB disruption and TNF transport into brain. We used the cecal ligation and puncture (CLP) model of sepsis in young and aged mice that were either undisturbed or had their sleep disrupted. There was a dichotomous effect of sepsis and sleep disruption with age: sepsis disrupted the BBB and increased TNF transport in young mice but not in aged mice, whereas sleep fragmentation disrupted the BBB and increased TNF transport in aged mice, but not in young mice. Combining sleep fragmentation and CLP did not produce a greater effect on either of these BBB parameters than did either of these manipulations alone. These results suggest that the mechanisms by which sleep fragmentation and sepsis alter BBB functions are fundamentally different from one another and that a major change in the organism's responses to those insults occurs with aging.

Recovery sleep does not mitigate the effects of prior sleep loss on paclitaxel-induced mechanical hypersensitivity in Sprague-Dawley rats.

Society has a rapidly growing accumulative sleep debt due to employment obligations and lifestyle choices that limit sleep opportunities. The degree to which poor sleep may set the stage for adverse symptom outcomes among more than 1.7 million persons who will be diagnosed with cancer is not entirely understood. Paclitaxel (PAC), a commonly used chemotherapy agent, is associated with painful, debilitating peripheral neuropathy of the hands and feet, which may persist long after adjuvant therapy is completed. The aims of this preclinical study were to determine the accumulative and sustained effects of sleep restriction on PAC-induced mechanical sensitivity in animals and whether there are male-female differences in mechanical sensitivity in PAC-injected animals. Sixty-two adult Sprague-Dawley rats (n = 31 females) were assigned to three cycles of intraperitoneal injections of PAC (1 mg/kg) versus vehicle (VEH; 1 ml/kg) every other day at light onset for 7 days, followed by seven drug-free days and to sleep restriction versus unperturbed sleep. Sleep restriction involved gentle handling to maintain wakefulness during the first 6 hr of lights on immediately following an injection; otherwise, sleep was unperturbed. Mechanical sensitivity was assessed via von Frey filaments, using the up-down method. Mechanical sensitivity data were Log10 transformed to meet the assumption of normality for repeated measures analysis of variance. Chronic sleep restriction of the PAC-injected animals resulted in significantly increased mechanical sensitivity that progressively worsened despite sleep recovery opportunities. If these relationships hold in humans, targeted sleep interventions employed during a PAC protocol may improve pain outcomes.

Acute increases in intramuscular inflammatory cytokines are necessary for the development of mechanical hypersensitivity in a mouse model of musculoskeletal sensitization.

Musculoskeletal pain is a widespread health problem in the United States. Back pain, neck pain, and facial pain are three of the most prevalent types of chronic pain, and each is characterized as musculoskeletal in origin. Despite its prevalence, preclinical research investigating musculoskeletal pain is limited. Musculoskeletal sensitization is a preclinical model of muscle pain that produces mechanical hypersensitivity. In a rodent model of musculoskeletal sensitization, mechanical hypersensitivity develops at the hind paws after injection of acidified saline (pH 4.0) into the gastrocnemius muscle. Inflammatory cytokines contribute to pain during a variety of pathologies, and in this study we investigate the role of local, intramuscular cytokines in the development of mechanical hypersensitivity after musculoskeletal sensitization in mice. Local intramuscular concentrations of interleukin-1ß (IL-1), IL-6 and tumor necrosis factor-α (TNF) were quantified following injection of normal (pH 7.2) or acidified saline into the gastrocnemius muscle. A cell-permeable inhibitor was used to determine the impact on mechanical hypersensitivity of inhibiting nuclear translocation of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) prior to musculoskeletal sensitization. The role of individual cytokines in mechanical hypersensitivity following musculoskeletal sensitization was assessed using knockout mice lacking components of the IL-1, IL-6 or TNF systems. Collectively, our data demonstrate that acidified saline injection increases intramuscular IL-1 and IL-6, but not TNF; that intramuscular pre-treatment with an NF-κB inhibitor blocks mechanical hypersensitivity; and that genetic manipulation of the IL-1 and IL-6, but not TNF systems, prevents mechanical hypersensitivity following musculoskeletal sensitization. These data establish that actions of IL-1 and IL-6 in local muscle tissue play an acute regulatory role in the development of mechanical hypersensitivity following musculoskeletal sensitization.

Sleep and immune function: glial contributions and consequences of aging.

The reciprocal interactions between sleep and immune function are well-studied. Insufficient sleep induces innate immune responses as evidenced by increased expression of pro-inflammatory mediators in the brain and periphery. Conversely, immune challenges upregulate immunomodulator expression, which alters central nervous system-mediated processes and behaviors, including sleep. Recent studies indicate that glial cells, namely microglia and astrocytes, are active contributors to sleep and immune system interactions. Evidence suggests glial regulation of these interactions is mediated, in part, by adenosine and adenosine 5'-triphosphate actions at purinergic type 1 and type 2 receptors. Furthermore, microglia and astrocytes may modulate declines in sleep-wake behavior and immunity observed in aging.

Sepsis-induced morbidity in mice: effects on body temperature, body weight, cage activity, social behavior and cytokines in brain.

Infection negatively impacts mental health, as evidenced by the lethargy, malaise, and cognitive deficits experienced during illness. These changes in central nervous system processes, collectively termed sickness behavior, have been shown in animal models to be mediated primarily by the actions of cytokines in brain. Most studies of sickness behavior to date have used bolus injection of bacterial lipopolysaccharide (LPS) or selective administration of the proinflammatory cytokines interleukin-1ß (IL-1ß) or IL-6 as the immune challenge. Such models, although useful for determining mechanisms responsible for acute changes in physiology and behavior, do not adequately represent the more complex effects on central nervous system (CNS) processes of a true infection with replicating pathogens. In the present study, we used the cecal ligation and puncture (CLP) model to quantify sepsis-induced alterations in several facets of physiology and behavior of mice. We determined the impact of sepsis on cage activity, body temperature, food and water consumption and body weights of mice. Because cytokines are critical mediators of changes in behavior and temperature regulation during immune challenge, we also quantified sepsis-induced alterations in cytokine mRNA and protein in brain during the acute period of sepsis onset. We now report that cage activity and temperature regulation in mice that survive are altered for up to 23 days after sepsis induction. Food and water consumption are transiently reduced, and body weight is lost during sepsis. Furthermore, sepsis decreases social interactions for 24-48 h. Finally, mRNA and protein for IL-1ß, IL-6, and tumor necrosis factor-α (TNFα) are upregulated in the hypothalamus, hippocampus, and brain stem during sepsis onset, from 6h to 72 h post sepsis induction. Collectively, these data indicate that sepsis not only acutely alters physiology, behavior and cytokine profiles in brain, but that some brain functions are impaired for long periods in animals that survive.

Ischemic stroke selectively inhibits REM sleep of rats.

Sleep disorders are important risk factors for stroke; conversely, stroke patients suffer from sleep disturbances including disruptions of non-rapid eye movement (NREM) and rapid eye movement (REM) sleep and a decrease in total sleep. This study was performed to characterize the effect of stroke on sleep architecture of rats using continuous electroencephalography (EEG) and activity monitoring. Rats were implanted with transmitters which enabled continuous real time recording of EEG, electromyography (EMG), and locomotor activity. Baseline recordings were performed prior to induction of either transient middle cerebral artery (MCA) occlusion or sham surgery. Sleep recordings were obtained for 60 h after surgery to identify periods of wakefulness, NREM, and REM sleep before and after stroke. Spectral analysis was performed to assess the effects of stroke on state-dependent EEG. Finally, we quantified the time in wake, NREM, and REM sleep before and after stroke. Delta power, a measure of NREM sleep depth, was increased the day following stroke. At the same time, there was a significant shift in theta rhythms to a lower frequency during REM and wake periods. The awake EEG slowed after stroke over both hemispheres. The EEG of the ischemic hemisphere demonstrated diminished theta power specific to REM in excess of the slowing seen over the contralateral hemisphere. In contrast to rats exposed to sham surgery which had slightly increased total sleep, rats undergoing stroke experienced decreased total sleep. The decrease in total sleep after stroke was the result of dramatic reduction in the amount of REM sleep after ischemia. The suppression of REM after stroke was due to a decrease in the number of REM bouts; the length of the average REM bout did not change. We conclude that after stroke in this experimental model, REM sleep of rats is specifically and profoundly suppressed. Further experiments using this experimental model should be performed to investigate the mechanisms and consequences of REM suppression after stroke.

Sepsis-induced alterations in sleep of rats.

Sepsis is a systemic immune response to infection that may result in multiple organ failure and death. Polymicrobial infections remain a serious clinical problem, and in the hospital, sepsis is the number-one noncardiac killer. Although the central nervous system may be one of the first systems affected, relatively little effort has been made to determine the impact of sepsis on the brain. In this study, we used the cecal ligation and puncture (CLP) model to determine the extent to which sepsis alters sleep, the EEG, and brain temperature (Tbr) of rats. Sepsis increases the amount of time rats spend in non-rapid eye movement sleep (NREMS) during the dark period, but not during the light period. Rapid eye movements sleep (REMS) of septic rats is suppressed for about 24 h following CLP surgery, after which REMS increases during dark periods for at least three nights. The EEG is dramatically altered shortly after sepsis induction, as evidenced by reductions in slow-frequency components. Furthermore, sleep is fragmented, indicating that the quality of sleep is diminished. Effects on sleep, the EEG, and Tbr persist for at least 84 h after sepsis induction, the duration of our recording period. Immunohistochemical assays focused on brain stem mechanisms responsible for alterations in REMS, as little information is available concerning infection-induced suppression of this sleep stage. Our immunohistochemical data suggest that REMS suppression after sepsis onset may be mediated, in part, by the brain stem GABAergic system. This study demonstrates for the first time that sleep and EEG patterns are altered during CLP-induced sepsis. These data suggest that the EEG may serve as a biomarker for sepsis onset. These data also contribute to our knowledge of potential mechanisms, whereby infections alter sleep and other central nervous system functions.

Sleep and fatigue in mice infected with murine gammaherpesvirus 68.

Fatigue, a common symptom of many acute and chronic medical conditions, reduces both quality of life and workplace productivity and can be disabling. However, the pathophysiologic mechanisms that underlie fatigue can be difficult to study in human populations due to the patient heterogeneity, the variety of underlying causes and potential triggering events, and an inability to collect samples that may be essential to elucidation of mechanisms (e.g., brain). Although the etiology of chronic fatigue syndrome (CFS) remains elusive, some studies have implicated viral infections, including Epstein-Barr virus (EBV), a human gammaherpesvirus, as a potential factor in the pathogenesis of CFS. Murine gammaherpesvirus 68 (γHV68) is a mouse pathogen that shares many similarities with human γHVs, including EBV. In this study, we use γHV68-infected C57BL/6J mice as a model system for studying the impact of chronic viral infection on sleep-wake behavior, activity patterns, and body temperature profiles. Our data show that γHV68 alters sleep, activity, and temperature in a manner suggestive of fatigue. In mice infected with the highest dose used in this study (40,000plaque forming units), food intake, body weight, wheel running, body temperature, and sleep were normal until approximately 7days after infection. These parameters were significantly altered during days 7 through 11, returned to baseline levels at day 12 after infection, and remained within the normal range for the remainder of the 30-day period after inoculation. At that time, both infected and uninfected mice were injected with lipopolysaccharide (LPS), and their responses monitored. Uninfected mice given LPS developed a modest and transient febrile response during the initial light phase (hours 12 through 24) after injection. In contrast, infected mice developed changes in core body temperatures that persisted for at least 5days. Infected mice showed an initial hypothermia that lasted for approximately 12h, followed by a modest fever that persisted for several hours. For the remainder of the 5-day recording period, they showed mild hypothermia during the dark phase. Running wheel activity of infected mice was reduced for at least 5days after injection of LPS, but for only 12h in uninfected mice. Collectively, these observations indicate that (1) physiologic and behavioral processes in mice are altered and recover during an early phase of infection, and (2) mice with latent γHV68 infection have an exacerbated response to challenge with LPS. These findings indicate that laboratory mice with γHV68 infections may provide a useful model for the study of fatigue and other physiologic and behavioral perturbations that may occur during acute and chronic infection with gammaherpesviruses.

Interleukin-1 inhibits putative cholinergic neurons in vitro and REM sleep when microinjected into the rat laterodorsal tegmental nucleus.

STUDY OBJECTIVES: REM sleep is suppressed during infection, an effect mimicked by the administration of cytokines such as interleukin-1 (IL-1). In spite of this observation, brain sites and neurochemical systems mediating IL-1-induced suppression of REM sleep have not been identified. Cholinergic neurons in the brainstem laterodorsal tegmental nucleus (LDT) are part of the neuronal circuitry responsible for REM sleep generation. Since IL-1 inhibits acetylcholine synthesis and release, the aim of this study was to test the two different, but related hypotheses. We hypothesized that IL-1 inhibits LDT cholinergic neurons, and that, as a result of this inhibition, IL-1 suppresses REM sleep. DESIGN, MEASUREMENT, AND RESULTS: To test these hypotheses, the electrophysiological activity of putative cholinergic LDT neurons was recorded in a rat brainstem slice preparation. Interleukin-1 significantly inhibited the firing rate of 76% of recorded putative cholinergic LDT neurons and reduced the amplitude of glutamatergic evoked potentials in 60% of recorded neurons. When IL-1 (1 ng) was microinjected into the LDT of freely behaving rats, REM sleep was reduced by about 50% (from 12.7% +/- 1.5% of recording time [after vehicle] to 6.1% +/- 1.4% following IL-1 administration) during post-injection hours 3-4. CONCLUSIONS: Results of this study support the hypothesis that IL-1 can suppress REM sleep by acting at the level of the LDT nucleus. Furthermore this effect may result from the inhibition of evoked glutamatergic responses and of spontaneous firing of putative cholinergic LDT neurons.

Two pathways for cyclooxygenase-2 protein degradation in vivo.

COX-2, formally known as prostaglandin endoperoxide H synthase-2 (PGHS-2), catalyzes the committed step in prostaglandin biosynthesis. COX-2 is induced during inflammation and is overexpressed in colon cancer. In vitro, an 18-amino acid segment, residues 595-612, immediately upstream of the C-terminal endoplasmic reticulum targeting sequence is required for N-glycosylation of Asn(594), which permits COX-2 protein to enter the endoplasmic reticulum-associated protein degradation system. To determine the importance of this COX-2 degradation pathway in vivo, we engineered a del595-612 PGHS-2 (Delta 18 COX-2) knock-in mouse lacking this 18-amino acid segment. Delta 18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. Delta 18 COX-2 mice do have elevated urinary prostaglandin E(2) metabolite levels and display a more pronounced and prolonged bacterial endotoxin-induced febrile response than wild type (WT) mice. Normal brain tissue, cultured resident peritoneal macrophages, and cultured skin fibroblasts from Delta 18 COX-2 mice overexpress Delta 18 COX-2 relative to WT COX-2 expression in control mice. These results indicate that COX-2 can be degraded via the endoplasmic reticulum-associated protein degradation pathway in vivo. Treatment of cultured cells from WT or Delta 18 COX-2 mice with flurbiprofen, which blocks substrate-dependent degradation, attenuates COX-2 degradation, and treatment of normal mice with ibuprofen increases the levels of COX-2 in brain tissue. Thus, substrate turnover-dependent COX-2 degradation appears to contribute to COX-2 degradation in vivo. Curiously, WT and Delta 18 COX-2 protein levels are similar in kidneys and spleens from WT and Delta 18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.

Lipopolysaccharide-induced increases in cytokines in discrete mouse brain regions are detectable using Luminex xMAP technology.

Methods to determine cytokine protein content in samples of interest, such as enzyme-linked immunosorbent assay (ELISA), are often labor-intensive and costly. Furthermore, because ELISA requires relatively large sample volumes and protein concentrations, it is difficult using this technique to determine protein content for multiple cytokines from individual samples. Recently, Luminex has developed an open source hardware platform combining flow cytometry- and bead-based antibody capture that is capable of detecting multiple analytes from a single sample. In the present study we employed the Luminex 200 platform to determine the cytokine protein content in discrete brain regions of C57BL/6J mice. In spike-and-recovery experiments, known concentrations of murine recombinant interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)alpha were added either singly or as a mixture of all three to whole brain homogenates containing known quantities of total protein. Spiked samples were assayed for either a single cytokine or for multiple cytokines using 1-plex or 3-plex assay kits, respectively. In whole mouse brain homogenate we recovered between 81% and 103% of the recombinant cytokines. We then injected C57BL/6J mice intraperitoneally with bacterial lipopolysaccharide (LPS) and sacrificed them 4h later. We detected in samples taken from LPS-stimulated mice 4- to 870-fold increases in serum or spleen cytokine protein, and 1.5- to 16-fold increases in cytokine protein in discrete brain regions, relative to protein content in samples obtained from vehicle-treated animals. These results indicate that multiple cytokines may be reliably assayed from discrete regions of mouse brain using a single sample.

Sleep-wake behavior and responses to sleep deprivation of mice lacking both interleukin-1 beta receptor 1 and tumor necrosis factor-alpha receptor 1.

Data indicate that interleukin (IL)-1 beta and tumor necrosis factor-alpha (TNFalpha) are involved in the regulation of non-rapid eye movement sleep (NREMS). Previous studies demonstrate that mice lacking the IL-1 beta type 1 receptor spend less time in NREMS during the light period, whereas mice lacking the p55 (type 1) receptor for TNFalpha spend less time in NREMS during the dark period. To further investigate roles for IL-1 beta and TNFalpha in sleep regulation we phenotyped sleep and responses to sleep deprivation of mice lacking both the IL-1 beta receptor 1 and TNFalpha receptor 1 (IL-1R1/TNFR1 KO). Male adult mice (IL-1R1/TNFR1 KO, n=14; B6129SF2/J, n=14) were surgically instrumented with EEG electrodes and with a thermistor to measure brain temperature. After recovery and adaptation to the recording apparatus, 48 h of undisturbed baseline recordings were obtained. Mice were then subjected to 6h sleep deprivation at light onset by gentle handling. IL-1R1/TNFR1 KO mice spent less time in NREMS during the last 6h of the dark period and less time in rapid eye movement sleep (REMS) during the light period. There were no differences between strains in the diurnal timing of delta power during NREMS. However, there were strain differences in the relative power spectra of the NREMS EEG during both the light period and the dark period. In addition, during the light period relative power in the theta frequency band of the REMS EEG differed between strains. After sleep deprivation, control mice exhibited prolonged increases in NREMS and REMS, whereas the duration of the NREMS increase was shorter and there was no increase in REMS of IL-1R1/TNFR1 KO mice. Delta power during NREMS increased in both strains after sleep deprivation, but the increase in delta power during NREMS of IL-1R1/TNFR1 KO mice was of greater magnitude and of longer duration than that observed in control mice. These results provide additional evidence that the IL-1 beta and TNFalpha cytokine systems play a role in sleep regulation and in the alterations in sleep that follow prolonged wakefulness.

Murine gammaherpesvirus 68: a model for the study of Epstein-Barr virus infections and related diseases.

Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus (GHV) that causes acute infection and establishes life-long latency. EBV is associated with the development of B-cell lymphoproliferative disorders, several malignant cancers, the syndrome of infectious mononucleosis, and chronic interstitial lung disease. Although the molecular biology of EBV has been characterized extensively, the associated disease conditions and their pathogenesis are difficult to study in human populations because of variation in human environments and genetics, the well-documented effect of stressors on pathogenesis, and the chronic and latent properties of the virus. GHV are highly species-specific, and suitable animal models for EBV are not available. However, in 1980, a murine gammaherpesvirus (MuGHV, also known as MHV68 and gammaHV68) was identified as a natural pathogen of bank voles and wood mice. Experimental MuGHV infections in laboratory mice share many features of EBV infections in humans, including facets of the clinical human syndrome known as infectious mononucleosis. These features make MuGHV a valuable experimental model for studying the pathophysiology of a GHV in a natural host.

Role of interleukin-6 in a glucan-induced model of granulomatous vasculitis.

The role of interleukin-6 (IL-6) in granulomatous vasculitis is not well understood. To investigate its involvement in this type of vasculitis a model of glucan-induced pulmonary vasculitis employed interleukin-6 deficient (IL-6-/-) mice. Briefly, IL-6-/- mice and C57B/J6 wild type (IL-6+/+) mice were injected intravenously with a suspension of glucan isolated from the cell wall of bakers yeast which results in a granulomatous vasculitis primarily in the pulmonary vasculature. Histological examination demonstrated no significant difference in the number of infiltrating leukocytes between the IL-6+/+ and IL-6-/- glucan-injured mice. Similar numbers of granulomas were noted in both the IL-6+/+ and IL-6-/- injured animals, while no granulomas were seen in saline injected control mice. Cells recovered from the bronchoalveolar lavage (BAL) fluid were differentially stained and counted. While there was a significant increase in infiltrating leukocytes recovered from the BAL following glucan-induced injury, there was no significant difference between the IL-6+/+ and IL-6-/- mice. In addition, no difference was demonstrated in total protein content in the BAL fluid between IL-6+/+ and IL-6-/- mice. However, myeloperoxidase (MPO) activity in the lungs of the IL-6-/- mice was less than in their IL-6+/+ counterparts suggesting that these animals have a partial defect in their ability to recruit neutrophils in this model. Studies done to look for levels of other cytokines/chemokines in these animals to compensate for the loss of IL-6 revealed that only IL-10 in the sera (p<0.016) and BAL fluid (p<0.05) of IL-6-/- mice was significantly higher then their IL-6+/+-injured counterparts. These studies suggest that IL-6, while possibly involved in early neutrophil accumulation in this model does not appear critical to the development of the TH-2 mediated granulomatous vasculitis.

Inhibition of caspase-1 in rat brain reduces spontaneous nonrapid eye movement sleep and nonrapid eye movement sleep enhancement induced by lipopolysaccharide.

Evidence suggests that IL-1beta is involved in promoting physiological nonrapid eye movement (NREM) sleep. IL-1beta has also been proposed to mediate NREM sleep enhancement induced by bacteria or their components. Mature and biologically active IL-1beta is cleaved from an inactive precursor by a cysteinyl aspartate-specific protease (caspase)-1. This study aimed to test the hypothesis that inhibition in brain of the cleavage of biologically active IL-1beta will reduce in rats both spontaneous NREM sleep and NREM sleep enhancement induced by the peripheral administration of components of the bacterial cell wall. To test this hypothesis, rats were intracerebroventricularly administered the caspase-1 inhibitor Ac-Tyr-Val-Ala-Asp chloromethyl ketone (YVAD; 3, 30, 300, and 1,500 ng) or were pretreated intracerebroventricularly with YVAD (300 ng) and then intraperitoneally injected with the gram-negative bacterial cell wall component LPS (250 microg/kg). Subsequent sleep-wake behavior was determined by standard polygraphic recordings. YVAD administration at the beginning of the light phase of the light-dark cycle significantly reduced time spontaneously spent in NREM sleep during the first 12 postinjection hours. YVAD pretreatment also completely prevented NREM sleep enhancement induced by peripheral LPS administration at the beginning of the dark phase. These results, in agreement with previous evidence, support the involvement of brain IL-1beta in physiological promotion of NREM sleep and in mediating NREM sleep enhancement induced by peripheral immune challenge.

Cytokines and normal sleep.

PURPOSE OF REVIEW: Cytokines are mediators of immune system responses with multiple biologic actions on several target tissues. Over the past two decades, research has explored the interactions between cytokines and sleep mechanisms of the brain. This short review highlights selected findings that have advanced our understanding of the relation between cytokines and sleep. RECENT FINDINGS: A complex network of cytokines and their receptors exists in brain. Cytokines may either promote or inhibit sleep. Of cytokines studied thus far, evidence indicates that interleukin-1 and tumor necrosis factor play a role in the regulation of non-rapid eye movement sleep. Their sites of action for regulating such sleep likely include the hypothalamic preoptic area and the basal forebrain. Mechanisms of action include direct receptor-mediated effects on neurons and the synthesis and release of numerous transmitters, peptides, and hormones that lead to subsequent changes in sleep. Among others, the cascade of responses induced by cytokines that may lead to subsequent alterations in sleep includes alterations in nitric oxide synthesis and effects on neurohormonal systems such as growth hormone releasing hormone. The activation by cytokines of the hypothalamic-pituitary-adrenal axis also influences sleep. Studies suggest that there is a significant overlap between neurohormonal systems such as the somatotropic and hypothalamic-pituitary-adrenal axes and cytokines, particularly with regard to their effects on sleep-wake regulation. SUMMARY: There is increasing evidence of a role for cytokines in regulating spontaneous non-rapid eye movement sleep. The somatotropic hormonal system and hypothalamic-pituitary-adrenal axis mediate, in part, the effects of cytokines on sleep.

Sleep-wake behavior and responses of interleukin-6-deficient mice to sleep deprivation.

Interleukin (IL)-1 and tumor necrosis factor (TNF) are involved in the regulation of non-rapid eye movements sleep (NREMS). Accumulating evidence suggests IL-6 modulates sleep under some pathophysiologic conditions. We used mice lacking a functional IL-6 gene to investigate further a potential role for IL-6 in the regulation of sleep. IL-6 knockout mice (B6.129S6-Il6tm1Kopf; n=10) and C57BL/6J mice (n=10) were purchased from the Jackson Laboratory (Bar Harbor, ME). Twenty-four-hour baseline recordings were obtained from mice in the absence of any experimental manipulation. Mice were then subjected to 6-h sleep deprivation beginning at light onset. Recordings were obtained during the deprivation period and for 18 h thereafter. During baseline conditions there were no differences between mouse strains with respect to the duration, timing or intensity of NREMS. However, across the 24-h recording period IL-6 knockout mice spent approximately 30% more time in rapid eye movements sleep (REMS) than did C57BL/6J mice. Relative to C57BL/6J mice, core body temperatures of IL-6 knockout mice were higher during the light period of the light:dark cycle. Both strains responded to sleep deprivation by spending more time in NREMS and REMS. Although the total increase in the amount of NREMS after sleep deprivation was the same in both strains, IL-6 knockout mice took 6h longer to accumulate this additional sleep. Under the conditions of this study, IL-6 does not appear necessary for the full manifestation of NREMS, although this cytokine may influence the dynamics of responses to sleep deprivation. That mice lacking IL-6 spend more time in REMS suggests that interactions between IL-6 and REMS regulatory mechanisms may differ from those of IL-1 and/or TNF.

Diurnal variation of lipopolysaccharide-induced alterations in sleep and body temperature of interleukin-6-deficient mice.

Infectious challenge triggers a broad array of coordinated changes within the host organism, including alterations in sleep-wake behavior and body temperature. Pro-inflammatory cytokines orchestrate many of the behavioral, metabolic, and endocrine responses to immune challenge. Although interleukin (IL)-6 mediates several aspects of sickness behavior, a role for this cytokine as a mediator of alterations in sleep in response to immune challenge has not been established. We evaluated sleep-wake behavior and core body temperature of IL-6-deficient (IL-6 KO; B6.129S6-Il6tm1Kopf) mice and C57BL/6J control mice after intraperitoneal (IP) administration of 10 microg lipopolysaccharide (LPS). Because feedback mechanisms that regulate responses to immune challenge exhibit circadian rhythms, we evaluated responses to LPS administered at the beginning of both the light and dark portions of the light:dark cycle. LPS-induced increases in non-rapid eye movements sleep (NREMS) of both mouse strains, but this increase was less pronounced in IL-6 KO mice than in C57BL/6J mice. Strain differences in LPS-induced increases in NREMS were greatest after light-onset administration. During the 12 h light period, NREMS of C57BL/6J mice increased from 53.0+/-1.7% of recording time after vehicle to 65.4+/-1.4% of recording time after LPS. During this same time period, NREMS of IL-6 KO mice increased from 50.5+/-1.8% after vehicle to only 52.4+/-1.8% of recording time after LPS. REMS of both mouse strains was suppressed to the same extent after LPS, irrespective of timing of administration. LPS-induced fever in C57BL/6J mice, with peak magnitude of 1.4+/-0.3 degrees C and 1.8+/-0.2 degrees C after dark onset and light onset administration, respectively. In contrast, this dose of LPS-induced profound hypothermia in IL-6 KO mice, with nadirs of hypothermia reaching 4.9+/-1.0 degrees C after injection at dark onset and 2.2+/-0.5 degrees C after administration at light onset. These results indicate that IL-6 mediates some of the effects of LPS on NREMS and body temperature of mice, and that the magnitude and duration of these effects differ as a function of the time at which the challenge is given.