Gene expression in lung and basal forebrain during influenza infection in mice.

Inbred mice develop strain-dependent changes in sleep during the first few days after inoculation with influenza virus. To identify genes with the potential to differentially modulate sleep under this condition, we performed complementary DNA microarray analysis of both lung and basal forebrain (BF) of infected (I) and uninfected BALB/cByJ (C) and C57BL/6J (B6) mice. This analysis showed significant variation in the expression pattern of 667 and 1217 of the surveyed genes in BF and lung, respectively (P < 0.01). Applying the additional criterion of an effect size >or=2, 495 genes differed in expression in lung compared with 204 in BF. In BF, more genes were differentially expressed as a function of mouse strain, whereas in lung, more genes were differentially expressed as a function of health status. Significant alterations in expression after infection were more numerous and robust in BALB/cByJ vs. C57BL/6J mice. Some genes showed significant variation in both tissues as a function of strain or condition, but the changes in general were not parallel. Genes that showed significant and robust variation as a function of strain, health status or tissue included those related to immune function, metabolism, signal transduction, cell cycle regulation, apoptosis and other miscellaneous categories. Different patterns of gene expression in BF of uninfected mice suggest the possibility of fundamental mechanistic differences in pathways that modulate vigilance in these strains, whereas differences in expression of lung of infected mice suggests different peripherally generated sleep-modulatory stimuli in the two strains.

Effect of environmental temperature on sleep, locomotor activity, core body temperature and immune responses of C57BL/6J mice.

Ambient temperature exerts a prominent influence on sleep. In rats and humans, low ambient temperatures generally impair sleep, whereas higher temperatures tend to promote sleep. The purpose of the current study was to evaluate sleep patterns and core body temperatures of C57BL/6J mice at ambient temperatures of 22, 26 and 30 degrees C under baseline conditions, after sleep deprivation (SD), and after infection with influenza virus. C57BL/6J mice were surgically implanted with electrodes for recording electroencephalogram (EEG) and electromyogram (EMG) and with intraperitoneal transmitters for recording core body temperature (T(c)) and locomotor activity. The data indicate that higher ambient temperatures (26 and 30 degrees C) promote spontaneous slow wave sleep (SWS) in association with reduced delta wave amplitude during SWS in C57BL/6J mice. Furthermore, higher ambient temperatures also promote recuperative sleep after SD. Thus, in mice, higher ambient temperatures reduced sleep depth under normal conditions, but augmented the recuperative response to sleep loss. Mice infected with influenza virus while maintained at 22 or 26 degrees C developed more SWS, less rapid eye movement sleep, lower locomotor activity and greater hypothermia than did mice maintained at 30 degrees C during infection. In addition, despite equivalent viral titers, mice infected with influenza virus at 30 degrees C showed less leucopenia and lower cytokine induction as compared with 22 and 26 degrees C, respectively, suggesting that less inflammation develops at the higher ambient temperature.

Reduced basal and lipopolysaccharide-stimulated adenosine A1 receptor expression in the brain of nuclear factor-kappaB p50-/- mice.

Adenosine promotes cytoprotection under conditions of infection, ischemic preconditioning and oxidative stress. Previous studies from our laboratory indicate that the expression of the adenosine A1 receptor (A1AR) is induced by oxidative stress via activation of nuclear factor (NF)-kappaB. The prototypic transcription factor is composed of homo- or heterodimers of p50 and p65 subunits. To determine the role of NF-kappaB in the regulation of the A1AR in vivo, we compared the A1AR RNA and protein levels in the brains of mice lacking the p50 subunit of NF-kappaB (p50-/- mice) and age-matched B6129PF2/J (F2) controls. Radioligand binding assays in the cortex revealed a significantly lower number of A(1)AR (maximal binding capacity, Bmax) in the cortex of p50-/- mice (151+/-62 fmol/mg protein) versus 479+/-181 fmol/mg protein in the F2 (N=5 per strain, P<0.05), but no change in the equilibrium dissociation constant. Similar reductions in A1AR were measured in the hippocampus, brain stem and hypothalamus and in peripheral tissues, such as the adrenal gland, kidney and spleen. Estimation of the A1AR following purification by antibody affinity columns also indicated reduced A1AR in the p50-/- mice cortex, as compared with the F2 mice. A1AR immunocytochemistry indicates distinct neuronal labeling in the F2 cortex, which was substantially reduced in similar sections obtained from p50-/- mice. The p50-/- mice expressed lower levels of A1AR mRNA than F2 mice, as determined by real time PCR. Quantitation of the A1AR transducing G proteins by Western blotting show significantly less Galphai3, no change in Galphai1, but higher levels of Galphao and Gbeta in the cortices of p50-/-, as compared with F2 mice. Administration of bacterial lipopolysaccharide (LPS), an activator of NF-kappaB, increased A1AR expression in the cortices of F2 mice but not p50-/- mice. Cortical neurons cultures prepared from p50-/- mice showed a greater degree of apoptosis, compared with neurons from F2 mice. Activation of the A1AR reduced apoptosis with greater efficacy in cultures from F2 than p50-/- mice. Taken together, these data support a role for NF-kappaB in determining both the basal and LPS-stimulated A1AR expression in vivo which could contribute to neuronal survival.

Spontaneous, homeostatic, and inflammation-induced sleep in NF-kappaB p50 knockout mice.

The dimeric transcription factor nuclear factor-kappaB (NF-kappaB) regulates several endogenous sleep-modulatory substances and thereby serves as a pivotal mediator of sleep-wake homeostasis. To further define the role of NF-kappaB in sleep regulation, we monitored sleep and temperature in mice that lack the p50 subunit of NF-kappaB [p50 knockout (KO) mice]. Compared with the control B6129PF2/J strain, p50 KO mice spend more time in slow-wave sleep (SWS) and rapid eye movement sleep (REMS) under normal conditions and show enhanced homeostatic recovery of sleep after sleep loss. p50 KO mice also show increased SWS and reduced REMS and temperature after the administration of lipopolysaccharide, yet they are behaviorally less responsive to challenge with influenza virus. These data support a role for NF-kappaB, and, in particular, for the p50 subunit, in the regulation of sleep in healthy mice and in mice experiencing immune challenge.

Strain differences in sleep patterns of healthy and influenza-infected inbred mice.

Influenza-infected C57BL/6J and BALB/cByJ mice respectively develop increased slow-wave sleep (SWS) during the dark phase and reduced SWS during the light phase of the 24 hour circadian cycle. To determine whether similar or alternative variations in SWS develop after influenza infection in other inbred strains of mice, we characterized the sleep patterns of additional strains both before and after influenza infection. Three strains (A/J, BALB/cByJ, and C3H/HeJ) showed light-phase SWS suppression, two strains (C57BL/6J and DBA/2J) showed dark-phase SWS enhancement, and one strain (A/J) showed dark-phase SWS suppression. Three strains (AKR/J, C57BR/cdJ, and FVB/NJ) did not show significant changes in SWS time on day two post-inoculation. Core temperatures were correlated to change in SWS time after infection, but were not correlated to SWS during the baseline period. These data support and expand the existing literature that indicates genetic modulation of sleep both in healthy mice and in mice undergoing viral infection.

Kissimmee River restoration: a case study.

Channelization of the Kissimmee River transformed a 167 km meandering river into a 9 metre deep, 75 metre wide, 90 km drainage canal (C-38) that is compartmentalized with levees and water control structures into a series of five stagnant pools. Channelization dramatically changed water level and flow characteristics, drained 21,000 hectares of floodplain wetlands and severely impacted fish and wildlife populations. A $500 million dollar restoration project will restore the ecological integrity of the river-floodplain system by reconstructing the natural river channel and reestablishing hydrologic processes. Sixty expectations have been established to quantify the ecosystem's recovery. The first phase of reconstruction was completed in February 2001 and included movement of 9.2 million cubic metres of earth to backfill 12 km of C-38, the explosive demolition of one water control structure, construction of two sections (2.4 km) of new river channel, and reestablishment of 24 contiguous km of river. Numerous social, political, and technical challenges have been encountered during the project's evolution. Recommendations are provided for future restoration projects.

Identifying genetic influences on sleep: an approach to discovering the mechanisms of sleep regulation.

Comparisons of sleep patterns of various inbred strains of mice have revealed differences in daily amounts of slow-wave sleep and rapid-eye movement sleep, in circadian patterns of sleep, and in some parameters of the electroencephalograms both in healthy mice and in mice undergoing microbial infections. Technical considerations will probably be an important variable in achieving consensus between different independent studies that use a genetic approach to identify sleep-regulatory genes or mechanisms. However, despite such differences, current data suggest that both normal sleep and various sleep disorders either have a genetic basis or are influenced by genetically determined physiologic or environmental predispositions. Excessive sleepiness, abnormal sleep patterns, nonrestorative sleep, and fatigue are becoming increasingly pervasive in modern society. Identifying genes that influence vigilance may ultimately contribute to a better understanding of the processes that control normal sleep and contribute to sleep disorders and may eventually promote the development of interventions to prevent or alleviate these disabling medical conditions.

Cytokine- and microbially induced sleep responses of interleukin-10 deficient mice.

Interleukin (IL)-1 and tumor necrosis factor (TNF) promote slow-wave sleep (SWS), whereas IL-10 inhibits the synthesis of IL-1 and TNF and promotes waking. We evaluated the impact of endogenous IL-10 on sleep-wake behavior by studying mice that lack a functional IL-10 gene. Under baseline conditions, C57BL/6-IL-10 knockout (KO) mice spent more time in SWS during the dark phase of the light-dark cycle than did genetically intact C57BL/6 mice. The two strains of mice showed generally comparable responses to treatment with IL-1, IL-10, or influenza virus, but differed in their responses to lipopolysaccharide (LPS). In IL-10 KO mice, LPS induced an initial transient increase and a subsequent prolonged decrease in SWS, as well as profound hypothermia. These responses were not observed in LPS-treated C57BL/6 mice. These data demonstrate that in the absence of endogenous IL-10, spontaneous SWS is increased and the impact of LPS on vigilance states is altered. Collectively, these observations support a role for IL-10 in sleep regulation and provide further evidence for the involvement of cytokines in the regulation of sleep.

Persistent transmission of mouse hepatitis virus by transgenic mice.

Variation in susceptibility to viral infection is well documented across mouse strains. Specific combinations of viral strains and murine hosts may favor viral infection and disease, and could potentially allow the unexpected development of chronic, persistent, or latent infections. In some genetically modified strains of mice, immune function and perhaps other physiologic or metabolic systems may be substantially or marginally impaired. In the case study reported here, we document the apparent persistent transmission of mouse hepatitis virus (MHV) over a two-year period by MHV-seropositive transgenic mice. Transmission occurred via direct contact with seropositive mice and exposure to contaminated bedding. However, MHV was not detected at diagnostic laboratories by use of viral isolation or reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of tissues from MHV-seropositive animals. Our observation, together with the constantly expanding varieties of immune-impaired or poorly characterized murine hosts and the burgeoning dissemination of these animals throughout the biomedical research community, indicate that unexpected pathophysiologic presentations of common murine viral diseases may present new challenges to the biomedical research community in the future.

Systemic bacterial invasion induced by sleep deprivation.

Profound sleep disruption in humans is generally believed to cause health impairments. Through comparative research, specific physical effects and underlying mechanisms altered by sleep deprivation are being elucidated. Studies of sleep-deprived animals previously have shown a progressive, chronic negative energy balance and gradual deterioration of health, which culminate in fatal bloodstream infection without an infectious focus. The present study investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of live microorganisms in body tissues that are normally sterile. The tissues cultured for microbial growth included the blood, four major organs, six regional lymph nodes, the intestine, and the skin. The principal finding was early infection of the mesenteric lymph nodes by bacteria presumably translocated from the intestine and bacterial migration to and transient infection of extraintestinal sites. Presence of pathogenic microorganisms and their toxins in tissues constitutes a septic burden and chronic antigenic challenge for the host. Bacterial translocation and pathogenic sequelae provide mechanisms by which sleep deprivation appears to adversely affect health.

Lipopolysaccharide effects on neuronal activity in rat basal forebrain and hypothalamus during sleep and waking.

Peripheral administration of lipopolysaccharide (LPS) is associated with alterations in sleep and the electroencephalogram. To evaluate potential neuronal mechanisms for the somnogenic effects of LPS administration, we used unanesthetized rats to survey the firing patterns of neurons in various regions of rat basal forebrain (BF) and hypothalamus during spontaneous sleep and waking and during the epochs of sleep and waking that occurred after the intraperitoneal administration of LPS. In the brain regions studied, LPS administration was associated with altered firing rates in 39% of the neurons examined. A larger proportion of LPS-responsive units showed vigilance-related alterations in firing rates compared with nonresponsive units. Approximately equal proportions of LPS-responsive neurons showed increased and decreased firing rates after LPS administration, with some units in the lateral preoptic area of the hypothalamus showing particularly robust increases. These findings are consistent with other studies showing vigilance-related changes in neuronal activity in various regions of BF and hypothalamus and further demonstrate that peripheral LPS administration alters neuronal firing rates in these structures during both sleep and waking.

Evaluation of an anesthetic regimen for retroorbital blood collection from mice.

Many Institutional Animal Care and Use Committees require anesthetization of mice for retroorbital blood collection procedures. Optimal anesthetic regimens should provide chemical restraint and adequate analgesia for a sufficient but otherwise minimal duration. This study was conducted to evaluate the effectiveness, safety, and practical utility of the general anesthetic combination of ketamine and medetomidine with and without the topical anesthetic proparacaine. In addition, we sought to evaluate the reversal of general anesthesia with atipamezole. The findings suggest that the combination of ketamine, medetomidine, proparacaine, and atipamezole is a safe and humane anesthetic regimen for retroorbital collection of blood samples from mice.

Toxicity evaluation of prophylactic treatments for mites and pinworms in mice.

The detection of external and internal parasites in laboratory mice is a particularly problematic aspect of animal health evaluation. Because these organisms must be detected by direct examination of the feces or hair coat, low-level infestation or sporadic shedding can make them difficult to detect, thereby undermining confidence that negative reports are truly negative. Prophylactic treatment of suspect colonies with anthelminthics and/or insecticides may therefore be indicated under some circumstances. However, when considering the use of prophylactic treatments, the potential for toxicity is an important factor, especially in genetically modified strains of mice. To evaluate the potential toxicity of prophylactic anti- parasitic treatments on strains of mice that are commonly used as experimental models and in genetic engineering in our facility, we surveyed a number of strains and ages of mice for toxic reactions during treatment regimens that combine anthelminthic and anti-acaricidal agents. Three experimental protocols (ivermectin, piperazine, and dichlorvos in combination; ivermectin alone; and fenbendazole/permethrin or fenbendazole/dichlorvos) were evaluated. Our data suggest a potential for toxicity associated with these treatments and indicate to us that prophylactic treatment regimens should be initiated with caution.

Food and water restriction protocols: physiological and behavioral considerations.

Food and water restriction protocols are common in animal research, yet they often elicit discussion and controversy among institutional animal care and use committee members who review them. Determining a single standard by which all restriction protocols can be evaluated or performed may not be realistic. However, information about the physiologic and behavioral impact of food and water restriction can provide a basis for making rational judgments about these issues in general. This review will discuss the physiologic and behavioral consequences of food or water deprivation periods of 24 h or less and of chronic restriction schedules, with special reference to protocols that use food or water restriction as a motivational tool for behavioral training.

An evaluation of analgesic regimens for abdominal surgery in mice.

This study was designed to evaluate the efficacy of several analgesic regimens for use after intraperitoneal implantation of telemetry transmitters in mice. The lengths of time required for postoperative recovery of food and water intake, locomotor activity, and core temperature of mice that did not receive postsurgical analgesic medication were compared to those of mice that were given either an analgesic in the drinking water or buprenorphine injections. Many measured variables were not substantially altered by analgesic medications. However, ibuprofen-treated mice demonstrated significantly greater locomotor activity on days 2 through 5 after surgery and a more rapid return to stable postsurgical levels of activity and water intake as compared to those in untreated mice. These changes are consistent with potential analgesic efficacy of the ibuprofen treatment regimen. Buprenorphine injections elicited hyperactivity, hyperthermia, and reduced food and water intake during both the immediate postsurgical recovery period and after apparent recuperation from surgery, as compared to effects observed in saline-treated mice. Evaluating the effect of analgesic regimens on postsurgical changes in physiologic and behavioral variables can be useful in assessing the efficacy of analgesic treatments, but some changes may indicate pharmacologic effects that do not reflect pain relief.

A quantitative genetic analysis of locomotor activity in CXB recombinant inbred mice.

Recent studies have identified genes that influence the length of the circadian period maintained by mice housed under constant lighting conditions. However, a less studied circadian activity variable is the amplitude of daily oscillations in locomotor activity. This parameter reflects spontaneous activity exhibited under standard lighting and housing conditions and, therefore, differs conceptually from assessments of exploratory or open-field activity, voluntary wheel-running, or circadian period during exposure to constant light or constant darkness conditions. We recently observed a greater daily amplitude of oscillation in spontaneous locomotor activity in C57BL/6 mice compared to BALB/cBy mice. To identify genetic loci with potential linkage to circadian variation in the amount of locomotor activity, we measured the spontaneous activity of 13 CXB recombinant inbred (RI) strains of mice. The probability density distributions of locomotor activity phenotypes for the 13 CXB RI strains were consistent with the presence of a low number of major quantitative trait loci affecting this trait. Regions of chromosomes 3, 8, 12, 13, and 19 showed provisional linkage to strain variation in locomotor activity. Probabilities of linkage were not sufficient for declaration of an activity-related quantitative trait locus but were sufficient to warrant further analysis either with additional RI strains or with F2 panels.

A quantitative genetic analysis of slow-wave sleep and rapid-eye movement sleep in CXB recombinant inbred mice.

Various inbred strains of mice show different daily amounts of slow-wave sleep (SWS) and rapid-eye movement sleep (REMS), suggesting the possibility of genetic influences on sleep propensity. Previous work by others studying the spontaneous sleep patterns of seven strains of CXB recombinant inbred (RI) mice suggested several candidate quantitative trait loci (QTLs) associated with variation in REMS. Extending this approach, we evaluated the sleep patterns of 13 CXB RI strains and conducted linkage analyses based on 223 discrete informative loci. The probability density distribution of light phase REMS for the CXB RI strains showed deflections that correspond approximately to the parental phenotypes. This type of pattern is consistent with the presence of a low number of major effect quantitative trait loci. Regions of chromosomes 4, 16, and 17 showed provisional linkage to strain variation in REMS. The distribution of loci further suggested that dark phase and light phase REMS may be regulated by different genetic factors. Probabilities of linkage were not sufficient for declaration of a quantitative trait locus for REMS but were sufficient to warrant further analysis either with additional RI strains or with F2 panels.

A quantitative genetic analysis of slow-wave sleep in influenza-infected CXB recombinant inbred mice.

Influenza-infected C57BL/6J mice spend increased amounts of time in slow-wave sleep (SWS) during the dark phase of the circadian cycle compared to healthy mice. In contrast, infected BALB/cByJ mice show a normal or reduced time in SWS, particularly during the light phase. To identify genetic loci with linkage to these traits, we measured sleep in 13 CXB recombinant inbred (RI) strains derived from a cross between C57BL/6ByJ and BALB/cByJ mice. The probability density distribution of sleep patterns of influenza-infected CXB RI mice showed modes that correspond roughly with the parental modes during the dark phase of the circadian cycle and are intermediate or C57BL/6-like during the light phase. These patterns are consistent with the presence of a low number of major effect quantitative trait loci (QTLs). Chromosomal regions with provisional association to strain variation in influenza-induced SWS patterns were identified. In particular, a 10- to 12-cM interval on Chr 6 between D6Mit74 and D6Mit188 contains a QTL (LRS = 16.6 at 1 cM proximal to D6Mit316; genomewide p < .05) that influences the SWS response to influenza infection during the light phase. We have provisionally named this QTL Srilp1 (sleep response to influenza, light phase 1). Candidate genes for mediation of this phenotype include Ghrhr (growth hormone releasing hormone receptor), Crhr2 (corticotropin releasing hormone receptor 2), and Cd8a (an epitope on cytotoxic T lymphocytes). Several other intervals achieved suggestive probability scores that are sufficient to warrant further analysis either with additional RI strains or with F2 panels. The analysis also suggests that dark phase and light phase responses are regulated by different genetic factors.

Effects of sleep deprivation and other stressors on the immune and inflammatory responses of influenza-infected mice.

Many stressors have well-documented effects on host immune competence. However, two important stressors that have not been extensively characterized in terms of their immune-modulatory properties are sleep deprivation and alterations in light:dark cycles. We therefore evaluated the effects of these stressors on the immune and inflammatory responses of mice inoculated intranasally with influenza virus. In contrast to a previous report, sleep deprivation did not significantly alter viral clearance or antibody titers of either virus-naive or immunized mice. Exposure to constant light also failed to affect these variables. However, repeated overnight restraint, a well-characterized stressor, reduced the pulmonary inflammatory response elicited by influenza virus, as previously reported. The data indicate that sleep deprivation and altered light cycles do not markedly influence selected host defense responses to influenza infection under the conditions tested.