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The reproducibility of pre-clinical research is an important concern that is now being voiced by constituencies that include the National Institutes of Health, the pharmaceutical industry, Congress, the public and the scientific community. An important facet of performing and publishing well-controlled reproducible pre-clinical research is to stabilize and more completely define the environment of the animal subjects. Scientists who use rodents in research generally recognize the importance of maintaining a stable animal environment. However, despite a theoretical and general awareness of these issues, many may lack a true appreciation of how significantly even seemingly minor variations in the environment can affect research outcomes. The purpose of this article is to help investigators gain a more comprehensive and substantiated understanding of the potentially significant impact of even seemingly minor environmental changes on the animals and the data. An important caveat to this article is that the examples presented were selected from a very large literature, admittedly in order to illustrate certain points. The goal of this article is not to provide an overview of the entire literature on how the environment affects rodents but rather to make preclinical scientists more aware of how these factors can potentially influence the experimental data and contribute to poor reproducibility of research. [ABSTRACT FROM AUTHOR]

This overview and data-based example indicate how large families of recombinant inbred ( RI) strains can be used to identify genetic loci and genes that underlie complex phenotypic differences among inbred mice. The RI approach requires no a priori expectations or assumptions about mechanisms that influence the phenotype, other than that variability is partly heritable. RI strains, which are produced by inbreeding the F2 progeny of two parental strains for at least 20 generations, have two major advantages. First, numerous subjects with identical genotypes can be analyzed to determine the average phenotype associated with that genotype, and second, it becomes practical to systematically accumulate large genome and phenome data sets for entire RI families, including sequence data, transcriptomes for many organs, and cell types and extensive data on gene-by-pathogen interactions. This enables the construction of far more sophisticated models of disease cause and progression. To illustrate the use of the systems genetics approach to infectious disease, we designed a simple study using three complementary families of RI strains ( CXB, BXD, and AXB/ BXA) that are differentially susceptible to intravenous challenge with the yeast Candida albicans. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) radicals are produced during normal cellular function, after tissue injury, and in response to immune system activation during infection. The transformation of NO to peroxynitrite is essential for mediating some of its physiological and/or cytotoxic actions. As the expression of the adenosine A1 receptor (A1AR) is regulated by oxidative stress, we evaluated the role of NO in the regulation of A1AR expression, a G protein-coupled receptor involved in cytoprotection in the central nervous system. Administration of the NO donor, S-nitrosylpenicillamine (SNAP), to pheochromocytoma 12 (PC12) cells increased A1AR protein in a time- and dose-dependent manner, with maximal induction observed with 20 lM SNAP at 24 h. The response to SNAP was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3 oxide (C-PTIO), and by the inhibition of nuclear factor-κB (NF-κB), implicating this transcription factor in the regulatory process. In addition SNAP also increased the degradation of Inhibitory κB-α (IκB-α), a marker of NF-κB activation. Furthermore, the induction of inducible nitric oxide synthase (iNOS) by lipopolysaccharide increased A1AR in PC12 cells and in mice, whereas the inhibition of NOS activity suppressed this response. We conclude that NO, via the activation of NF-κB, serves as an endogenous regulator of A1AR, and speculate that the induction of the A1AR could counteract the cytotoxicity of NO. [ABSTRACT FROM AUTHOR]

Mice develop changes in sleep during the nonspecific immune response that occurs during the initial few days after inoculation with influenza virus. T lymphocytes, neutrophils, macrophages, and natural killer (NK) cells all participate in the early host response to influenza infection. All of these cell types are potential sources of endogenous substances that modulate sleep, but the contributory role of each cell type to the alteration of somnolence during infection has not been determined. To investigate which cell types contribute to the sleep enhancement that develops during influenza infection in mice, the sleep patterns of C57BL/6J mice with perturbations of particular facets of host immune response capabilities were assessed before and after influenza infection. Targeted mutation of the gene Ccl3 (macrophage inflammatory protein 1α)) prevented development of the dark phase sleep enhancement that is characteristic of C57BL/6J mice after influenza infection. Other experimental treatments that impair macrophage or monocyte function also produced significant (administration of pentoxifylline or CNI-1493) or marginally significant (deletion of the interferon-gamma gene or intranasal administration of carrageenan) changes in influenza-induced sleep enhancement in C57BL/6J mice. In contrast, functional impairments of NK cells, neutrophils, and T lymphocytes did not significantly influence sleep responses. These data therefore support a contributory role for macrophages, but not for NK cells, neutrophils, and T lymphocytes, in eliciting the sleep response typical of influenza-infected C57BL/6J mice. [Copyright &y& Elsevier]

With rapidly increasing enrollment and soaring tuition becoming major concerns of students, instructors, and administrators in many institutions of higher learning, the search to identify the most effective ways of delivering a high quality education at a reasonable cost continues. The decrease in government funding affecting many state-owned colleges and universities nationwide has often resulted in a decrease of course sections offered and an increase in the class size of the remaining available classes. The purpose of this review is to examine the current research regarding the relationship of class size to student achievement in higher education. The eight studies examined, published between 1990 and 2000, were analyzed as to methodological strength, measurement of achievement, significant findings, and resulting recommendations. The studies yielded mixed results, and upon close examination, it was found that the design methodologies were weak overall, with four of the eight studies examined defining “achievement” as simply the students' class grade alone. It is suggested that future research in this area investigate the possibility that other variables such as potential grade inflation, lower academic standards, student aptitude and readiness for college work, lack of remediation for ill-prepared and disadvantaged students, student learning styles, instructors' teaching styles, and student motivation and effort could confound research results in this area and may account for the inconsistent results. [ABSTRACT FROM AUTHOR]

Features a study on mice that lack of a functional interleukin (IL)-10 gene to evaluate the impact of endogeneous IL-10 on sleep-wake behavior. Background of the study; Materials and methods; Results.

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. [ABSTRACT FROM AUTHOR]

Presents a study which investigated the conditions antecedent to advanced morbidity in sleep-deprived rats by determining the time course and distribution of microorganisms in body tissues. Methodology of the study; Discussion on the results of the study; Conclusions.

Investigates 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. Mechanisms by which sleep deprivation appears to adversely affect health; Bacterial translocation and pathogenic sequelae.

Discusses the use of unanesthetized rats to survey the firing patterns of neurons in various regions of rat basal forebrain (BF) and hypothalamus during spontaneous sleep and waking. Evaluation of the potential neuronal mechanisms for the somnogenic effects of lipopolysaccharide administration; Changes in neuronal activity in regions of BF and hypothalamus.

Focuses on a study that aimed to evaluate the potential neuronal mechanisms for the somnogenic effects of lipopolysaccharide administration in rat basal forebrain and hypothalamus during sleep and waking. Methodology of the study; Results and discussion; Conclusion.

Determines whether slow-wave activity correlates with arousability in rabbits. Measures of arousability used; Increase in behavioral responsiveness to auditory stimuli with increasing stimulus intensity; Fewer postural changes exhibited by sleep-deprived animals; Correlation of latency to return to sleep with delta power before stimulus presentation.

Explores the effect of cluster course teaching on student academic achievement, self-esteem, sense of alienation and perceived meaningfulness of the college experience. No measurable benefits found for the college freshmen in the study.

Examines whether slow-wave activity in the electroencephalogram reflects the depth or intensity of sleep, using an auditory stimuli of 72-90 dB, to control or sleep-deprived rabbits. Influence of prior waking on arousability; Behavioral responses and the latency to return to sleep as dependent measures of arousability from sleep.

Evaluates the influence of light on sleep patterns after microbial challenge. Subjects and experimental design; Electrophysiological recordings; Fungal inocula and blood analysis; Statistical analysis.

Compares acute phase responses induced in rabbits by microbial lipopolysaccharide and double-stranded RNA. Systemic and central reactions from infection or injury; Luecocyte number; Body temperature; Antiviral activity; Ceruloplasmin; Fibrinogen.

Highlight: [•] After environmental perturbation, mice show systemic inflammation and reduced activity, in part independent of mouse strain and infection status. [ABSTRACT FROM AUTHOR]

Abstract: Many studies of influenza severity have focused on viral properties that confer virulence, whereas the contributory role of the host genetic background on infection severity remains largely unexplored. In this study, we measure the impact of inoculation with influenza virus in four strains of inbred mice - BALB/cByJ, C57BL/6J, A/J, and DBA/2J. To evaluate the extent to which responses are inherent to lung per se, as opposed to effects of the systemic response to lung infection, we also measured cytokines and chemokines in lung slices exposed to the virus in vitro. Finally, we evaluate the in vivo responses of recombinant inbred (RI) and select consomic strains of mice to search for genomic loci that contribute to phenotypic variance in response to influenza infection. We found marked variation among mouse strains after challenge with virus strain A/HKX31(H3N2), consistent with previous reports using more virulent strains. Furthermore, response patterns differ after in vivo versus in vitro exposure of lung to virus, supporting a predominant role of the systemic host inflammatory response in generating the strain differences. These results add to the body of information pointing to host genotype as a crucial factor in mediating the severity of influenza infections. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Aims: The type 2 muscarinic receptor (M2R) differs from the other G-protein-coupled muscarinic receptor (type 4, or M4R) in tissue distribution and physiologic effects. We studied the impact of these receptors on sleep and arousal by using M2R and M4R knock-out (KO) mice. Main methods: M2R and M4R KO and genetically intact mice were compared in terms of normal patterns of sleep, responses to sleep loss, infectious challenge and acoustic startle, and acoustic prepulse inhibition of startle (PPI). Key findings: Under basal conditions, M2R and M4R KO mice do not differ from the background strain or each other in the amount or diurnal pattern of sleep, locomotor activity, and body temperature. After enforced sleep loss, M2R KO mice, in contrast to the other two strains, show no rebound in slow-wave sleep (SWS) time, although their SWS is consolidated, and they show a greater rebound in time spent in REMS (rapid-eye-movement sleep) and REMS consolidation. During influenza infection, M2R KO mice, as compared with the other strains, show marked hypothermia and a less robust increase in SWS. During Candida albicans infection, M2R KO mice show a greater increase in SWS and a greater inflammatory response than do the other strains. M2R KO mice also show greater acoustic startle amplitude than does the background strain, although PPI was not different across the 3 strains over a range of stimulus intensities. Significance: Taken together, these findings support different roles for M2R and M4R in the modulation of sleep and arousal during homeostatic challenge. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Ginseng is a widely consumed aromatic herb that is purported to have health benefits. Several studies report a beneficial impact of ginseng or its derivatives on Candida albicans infection in mice and suggest that its immune-modulatory properties contribute to this effect. However, these studies generally administered ginseng to experimental animals by injection, whereas people typically ingest ginseng. Furthermore, although disseminated candiasis is typically a disease of immune-impaired hosts, previous studies have generally used immune competent host species in the assessments. Materials and methods: We evaluated the efficacy of an ingested extract of ginseng against Candida albicans infection in DBA/2J mice, which are highly susceptible to Candida albicans infection. A ginseng extract was added to the drinking water for two days before and for the remainder of the study after intravenous inoculation of mice with Candida albicans. Mice were evaluated for morbidity, mortality, Candida albicans titers, and concentrations of inflammatory cytokines and chemokines. Results: Ingestion of the ginseng extract did not significantly affect overall morbidity or mortality. However, ingestion of the extract was associated with significantly lower renal titers of Candida albicans and with significantly lower concentrations of some inflammatory cytokines in kidney and/or serum. Conclusions: Assessment of morbidity, mortality, inflammatory markers, and renal titers after spontaneous ingestion of ginseng by susceptible hosts represents a comprehensive approach to characterizations of therapeutic efficacy against infectious agents. Our findings extend previous reports of the efficacy of ginseng against Candida albicans by demonstrating significant reductions in infectious load and some markers of inflammation in susceptible mice. Our data therefore support further assessment of the immune-modulatory properties of this widely consumed herb and its components. [Copyright &y& Elsevier]

Abstract: Aims: Our previous work revealed that mice lacking the p50 subunit of transcription factor nuclear factor kappa B (NF-κB) (p50 KO mice) and genetically intact F2 mice have similar locomotion under basal conditions, yet p50 KO mice show greater locomotor activation after caffeine ingestion. In this report, we test whether KO mice display altered caffeine pharmacokinetics or increased caffeine-induced DA turnover relative to F2 mice, and evaluate the impact of intraperitoneal administration of specific adenosine and DA receptor antagonists on locomotor activity. Main methods: Concentrations of DA and caffeine were measured using high performance liquid chromatography. DA turnover was measured after treatment of mice with an inhibitor of tyrosine hydroxylase. Locomotor activity was measured using telemetry. Key findings: The data reveal that 1) caffeine concentrations in blood and brain are similar in KO and F2 mice after oral or intraperitoneal administration; 2) KO mice show greater DA turnover under basal conditions, but turnover is similar in both strains after caffeine administration; 3) the specific A2AAR antagonist SCH 58261 induces greater locomotion in KO versus F2 mice; and 4) the activating effect of SCH 58261 in KO mice is prevented by prior treatment with the D2R antagonist raclopride. Significance: These findings support the conclusions that 1) A2AAR has a major impact on behavioral activation of p50 KO mice, and 2) D2R mediated neurotransmission is important to this effect. [Copyright &y& Elsevier]

Abstract: The striatal dopamine D2 receptor (D2R) and adenosine A2A receptor (A2AAR) exhibit mutually antagonistic effects through physical interactions and by differential modulation of post-receptor signaling pathways. The expression of the A2AAR and the D2R is differentially regulated by nuclear factor-κB (NF-κB). In this report, we determined the role of NF-κB in regulation of these receptors by comparing mice deficient in the NF-κB p50 subunit (p50 KO) with genetically intact B6129PF2/J (F2) mice. Quantification of adenosine receptor (AR) subtypes in mouse striatum by real time PCR, immunocytochemistry and radioligand binding assays showed more A2AAR but less A1AR in p50 KO mice as compared with F2 mice. Striata from p50 KO mice also had less D2R mRNA and [3H]-methylspiperone binding than did striata from F2 mice. Gαolf and Gαs proteins, which are transducers of A2AAR signals, were also present at a higher level in striata from the p50 KO versus F2 mice. In contrast, the Gαi1 protein, which transduces signals from the A1AR and D2R, was significantly reduced in striata from p50 KO mice. Behaviorally, p50 KO mice exhibited increased locomotor activity relative to that of F2 mice after caffeine ingestion. These data are consistent with a role for the NF-κB in the regulation of A1AR, A2AAR, D2R and possibly their coupling G proteins in the striatum. Dysregulation of these receptors in the striata of p50 KO mice might sensitize these animals to locomotor stimulatory action of caffeine. [Copyright &y& Elsevier]