Chronotherapeutics for Solid Tumors.

Circadian rhythms are internal manifestations of the 24-h solar day that allow for synchronization of biological and behavioral processes to the external solar day. This precise regulation of physiology and behavior improves adaptive function and survival. Chronotherapy takes advantage of circadian rhythms in physiological processes to optimize the timing of drug administration to achieve maximal therapeutic efficacy and minimize negative side effects. Chronotherapy for cancer treatment was first demonstrated to be beneficial more than five decades ago and has favorable effects across diverse cancer types. However, implementation of chronotherapy in clinic remains limited. The present review examines the evidence for chronotherapeutic treatment for solid tumors. Specifically, studies examining chrono-chemotherapy, chrono-radiotherapy, and alternative chronotherapeutics (e.g., hormone therapy, TKIs, antiangiogenic therapy, immunotherapy) are discussed. In addition, we propose areas of needed research and identify challenges in the field that remain to be addressed.

Photoperiod regulates hypothalamic miR-155 gene expression in female, but not male, Siberian hamsters (Phodopus sungorus).

In many species, seasonal changes in photoperiod regulate several behaviors and physiological systems, including reproduction, energy balance, and immune function. MicroRNAs (miRs) regulate numerous physiological processes and developmental transitions through translational repression and mRNA degradation. Their role in seasonal transitions has been vastly understudied, with only a few reports in animals. Furthermore, no study has assessed whether there are sex differences in seasonal regulation of miRs. miR-155 is a primary candidate for seasonal regulation because it influences immune responses, energetics, and reproductive function. In this study, we tested the hypothesis that photoperiod regulates miR-155 gene expression in Siberian hamsters and whether there were sex differences in this photoperiod regulation. miR-155 gene expression levels were measured in hypothalamus, hippocampus, and spleen of male and female Siberian hamsters reared in short days (SDs) or long days (LDs). As expected, SD-reared hamsters had significantly reduced body mass, lightened pelage color, and lower reproductive organ size than LD-reared hamsters. Notably, SDs increased hypothalamic miR-155 gene expression in females but not in males. No differences were observed in hippocampus and spleen of either sex. These findings demonstrate sex-specific photoperiod regulation of miR-155 gene expression. Future studies should consider possible sex differences in miR contributions to seasonal changes in physiology and behavior. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Time-of-Day Dictates Transcriptional Inflammatory Responses to Cytotoxic Chemotherapy.

Many cytotoxic chemotherapeutics elicit a proinflammatory response which is often associated with chemotherapy-induced behavioral alterations. The immune system is under circadian influence; time-of-day may alter inflammatory responses to chemotherapeutics. We tested this hypothesis by administering cyclophosphamide and doxorubicin (Cyclo/Dox), a common treatment for breast cancer, to female BALB/c mice near the beginning of the light or dark phase. Mice were injected intravenously with Cyclo/Dox or the vehicle two hours after lights on (zeitgeber time (ZT2), or two hours after lights off (ZT14). Tissue was collected 1, 3, 9, and 24 hours later. Mice injected with Cyclo/Dox at ZT2 lost more body mass than mice injected at ZT14. Cyclo/Dox injected at ZT2 increased the expression of several pro-inflammatory genes within the spleen; this was not evident among mice treated at ZT14. Transcription of enzymes within the liver responsible for converting Cyclo/Dox into their toxic metabolites increased among mice injected at ZT2; furthermore, transcription of these enzymes correlated with splenic pro-inflammatory gene expression when treatment occurred at ZT2 but not ZT14. The pattern was reversed in the brain; pro-inflammatory gene expression increased among mice injected at ZT14. These data suggest that inflammatory responses to chemotherapy depend on time-of-day and are tissue specific.

Acute melatonin treatment alters dendritic morphology and circadian clock gene expression in the hippocampus of Siberian hamsters.

In the hippocampus of Siberian hamsters, dendritic length and dendritic complexity increase in the CA1 region whereas dendritic spine density decreases in the dentate gyrus region at night. However, the underlying mechanism of the diurnal rhythmicity in hippocampal neuronal remodeling is unknown. In mammals, most daily rhythms in physiology and behaviors are regulated by a network of circadian clocks. The central clock, located in the hypothalamus, controls melatonin secretion at night and melatonin modifies peripheral clocks by altering expression of circadian clock genes. In this study, we examined the effects of acute melatonin treatment on the circadian clock system as well as on morphological changes of hippocampal neurons. Male Siberian hamsters were injected with melatonin in the afternoon; 4 h later, mRNA levels of hypothalamic and hippocampal circadian clock genes and hippocampal neuron dendritic morphology were assessed. In the hypothalamus, melatonin treatment did not alter Period1 and Bmal1 expression. However, melatonin treatment increased both Period1 and Bmal1 expression in the hippocampus, suggesting that melatonin affected molecular oscillations in the hippocampus. Melatonin treatment also induced rapid remodeling of hippocampal neurons; melatonin increased apical dendritic length and dendritic complexity in the CA1 region and reduced the dendritic spine density in the dentate gyrus region. These data suggest that structural changes in hippocampal neurons are regulated by a circadian clock and that melatonin functions as a nighttime signal to coordinate the diurnal rhythm in neuronal remodeling.

Cytotoxic chemotherapy increases sleep and sleep fragmentation in non-tumor-bearing mice.

Sleep disruption ranks among the most common complaints of breast cancer patients undergoing chemotherapy. Because of the complex interactions among cancer, treatment regimens, and life-history traits, studies to establish a causal link between chemotherapy and sleep disruption are uncommon. To investigate how chemotherapy acutely influences sleep, adult female c57bl/6 mice were ovariectomized and implanted with wireless biotelemetry units. EEG/EMG biopotentials were collected over the course of 3days pre- and post-injection of 13.5mg/kg doxorubicin and 135mg/kg cyclophosphamide or the vehicle. We predicted that cyclophosphamide+doxorubicin would disrupt sleep and increase central proinflammatory cytokine expression in brain areas that govern vigilance states (i.e., hypothalamus and brainstem). The results largely support these predictions; a single chemotherapy injection increased NREM and REM sleep during subsequent active (dark) phases; this induced sleep was fragmented and of low quality. Mice displayed marked increases in low theta (5-7Hz) to high theta (7-10Hz) ratios following chemotherapy treatment, indicating elevated sleep propensity. The effect was strongest during the first dark phase following injection, but mice displayed disrupted sleep for the entire 3-day duration of post-injection sleep recording. Vigilance state timing was not influenced by treatment, suggesting that acute chemotherapy administration alters sleep homeostasis without altering sleep timing. qPCR analysis revealed that disrupted sleep was accompanied by increased IL-6 mRNA expression in the hypothalamus. Together, these data implicate neuroinflammation as a potential contributor to sleep disruption after chemotherapy.

Central IKKß inhibition prevents air pollution mediated peripheral inflammation and exaggeration of type II diabetes.

BACKGROUND: Prior experimental and epidemiologic data support a link between exposure to fine ambient particulate matter (<2.5 µm in aerodynamic diameter, PM2.5) and development of insulin resistance/Type II diabetes mellitus (Type II DM). We investigated the role of hypothalamic inflammation in PM2.5-mediated diabetes development. METHODS: KKay mice, a genetically susceptible model of Type II DM, were assigned to either concentrated PM2.5 or filtered air (FA) for 4-8 weeks via a versatile aerosol concentrator and exposure system, or administered intra-cerebroventricular with either IKKß inhibitor (IMD-0354) or TNFα antibody (infliximab) for 4-5 weeks simultaneously with PM2.5 exposure. Glucose tolerance, insulin sensitivity, oxygen consumption and heat production were evaluated. At euthanasia, blood, spleen, visceral adipose tissue and hypothalamus were collected to measure inflammatory cells using flow cytometry. Standard immunohistochemical methods and quantitative PCR were used to assess targets of interest. RESULTS: PM2.5 exposure led to hyperglycemia and insulin resistance, which was accompanied by increased hypothalamic IL-6, TNFα, and IKKß mRNA expression and microglial/astrocyte reactivity. Targeting the NFκB pathway with intra-cerebroventricular administration of an IKKß inhibitor [IMD-0354, n = 8 for each group)], but not TNFα blockade with infliximab [(n = 6 for each group], improved glucose tolerance, insulin sensitivity, rectified energy homeostasis (O2 consumption, CO2 production, respiratory exchange ratio and heat generation) and reduced peripheral inflammation in response to PM2.5. CONCLUSIONS: Central inhibition of IKKß prevents PM2.5 mediated peripheral inflammation and exaggeration of type II diabetes. These results provide novel insights into how air pollution may mediate susceptibility to insulin resistance and Type II DM.

Sleep deprivation attenuates endotoxin-induced cytokine gene expression independent of day length and circulating cortisol in male Siberian hamsters (Phodopus sungorus).

Sleep is restorative, whereas reduced sleep leads to negative health outcomes, such as increased susceptibility to disease. Sleep deprivation tends to attenuate inflammatory responses triggered by infection or exposure to endotoxin, such as bacterial lipopolysaccharide (LPS). Previous studies have demonstrated that Siberian hamsters (Phodopus sungorus), photoperiodic rodents, attenuate LPS-induced fever, sickness behavior and upstream pro-inflammatory gene expression when adapted to short day lengths. Here, we tested whether manipulation of photoperiod alters the suppressive effects of sleep deprivation upon cytokine gene expression after LPS challenge. Male Siberian hamsters were adapted to long (16 h:8 h light:dark) or short (8 h:16 h light:dark) photoperiods for >10 weeks, and were deprived of sleep for 24 h using the multiple platform method or remained in their home cage. Hamsters received an intraperitoneal injection of LPS or saline (control) 18 h after starting the protocol, and were killed 6 h later. LPS increased liver and hypothalamic interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF) gene expression compared with vehicle. Among LPS-challenged hamsters, sleep deprivation reduced IL-1 mRNA levels in liver and hypothalamus, but not TNF. IL-1 attenuation was independent of circulating baseline cortisol, which did not increase after sleep deprivation. Conversely, photoperiod altered baseline cortisol, but not pro-inflammatory gene expression in sleep-deprived hamsters. These results suggest that neither photoperiod nor glucocorticoids influence the suppressive effect of sleep deprivation upon LPS-induced inflammation.

Photoperiod alters fear responses and basolateral amygdala neuronal spine density in white-footed mice (Peromyscus leucopus).

Photoperiodism is a biological phenomenon in which environmental day length is monitored to ascertain time of year to engage in seasonally appropriate adaptations. This trait is common among organisms living outside of the tropics. White-footed mice (Peromyscus leucopus) are small photoperiodic rodents which display a suite of adaptive responses to short day lengths, including reduced hippocampal volume, impairments in hippocampal-mediated memory, and enhanced hypothalamic-pituitary-adrenal axis reactivity. Because these photoperiodic changes in brain and behavior mirror some of the etiology of post-traumatic stress disorder (PTSD), we hypothesized that photoperiod may also alter fear memory and neuronal morphology within the hippocampus-basolateral amygdala-prefrontal cortex fear circuit. Ten weeks of exposure to short days increased fear memory in an auditory-cued fear conditioning test. Short days also increased dendritic spine density of the neurons of the basolateral amygdala, without affecting morphology of pyramidal neurons within the infralimbic region of the medial prefrontal cortex. Taken together, photoperiodic phenotypic changes in brain morphology and physiology induced by a single environmental factor, exposure to short day lengths, affect responses to fearful stimuli in white-footed mice. These results have potential implications for understanding seasonal changes in fear responsiveness, as well as for expanding translational animal models for studying gene-environment interactions underlying psychiatric diseases, such as PTSD.

Dietary arginine depletion reduces depressive-like responses in male, but not female, mice.

Previous behavioral studies have manipulated nitric oxide (NO) production either by pharmacological inhibition of its synthetic enzyme, nitric oxide synthase (NOS), or by deletion of the genes that code for NOS. However manipulation of dietary intake of the NO precursor, L-arginine, has been understudied in regard to behavioral regulation. L-Arginine is a common amino acid present in many mammalian diets and is essential during development. In the brain L-arginine is converted into NO and citrulline by the enzyme, neuronal NOS (nNOS). In Experiment 1, paired mice were fed a diet comprised either of an L-arginine-depleted, L-arginine-supplemented, or standard level of L-arginine during pregnancy. Offspring were continuously fed the same diets and were tested in adulthood in elevated plus maze, forced swim, and resident-intruder aggression tests. L-Arginine depletion reduced depressive-like responses in male, but not female, mice and failed to significantly alter anxiety-like or aggressive behaviors. Arginine depletion throughout life reduced body mass overall and eliminated the sex difference in body mass. Additionally, arginine depletion significantly increased corticosterone concentrations, which negatively correlated with time spent floating. In Experiment 2, adult mice were fed arginine-defined diets two weeks prior to and during behavioral testing, and again tested in the aforementioned tests. Arginine depletion reduced depressive-like responses in the forced swim test, but did not alter behavior in the elevated plus maze or the resident intruder aggression test. Corticosterone concentrations were not altered by arginine diet manipulation in adulthood. These results indicate that arginine depletion throughout development, as well as during a discrete period during adulthood ameliorates depressive-like responses. These results may yield new insights into the etiology and sex differences of depression.

Short photoperiods alter cannabinoid receptor expression in hypothalamic nuclei related to energy balance.

This study examined the photoperiodic regulation of energy balance and cannabinoid receptor expression in the Siberian hamster (Phodopus sungorus) hypothalamus. Short day lengths, beginning at weaning, reduced food intake, body mass and fat pad masses and also decreased cannabinoid receptor immunostaining in the anterior and lateral hypothalamic nuclei of male hamsters. These data suggest a potential role for reduced cannabinoid drive in mediating short day-induced alterations in energy balance.

Estrous phase alters social behavior in a polygynous but not a monogamous Peromyscus species.

The social organization of rodent species determines behavioral patterns for both affiliative and agonistic encounters. The neuropeptide oxytocin has been implicated in the mediation of social behavior; however, variability in both neuropeptide expression and social behavior within a single species indicates an additional mediating factor. The purpose of the present comparative study was to investigate social behaviors in naïve mixed-sex pairs of monogamous Peromyscus californicus and polygynous Peromyscus leucopus. We identified substantial inter- and intra-specific variability in the expression of affiliative and agonistic behaviors. Although all P. californicus tested engaged in frequent and prolonged intervals of social contact and rarely engaged in aggressive behaviors, P. leucopus exhibited significant variability in both measures of social behaviors. The naturally occurring differences in social behavior displayed by P. leucopus vary across the estrous cycle, and correspond to hypothalamic oxytocin, as well as circulating oxytocin and glucocorticoid concentrations. These results provide evidence for a rhythm in social behavior across the estrous cycle in polygynous, but not monogamous, Peromyscus species.

Stressor-specific alterations in corticosterone and immune responses in mice.

Different stressors likely elicit different physiological and behavioral responses. Previously reported differences in the effects of stressors on immune function may reflect qualitatively different physiological responses to stressors; alternatively, both large and subtle differences in testing protocols and methods among laboratories may make direct comparisons among studies difficult. Here we examine the effects of chronic stressors on plasma corticosterone concentrations, leukocyte redistribution, and skin delayed-type hypersensitivity (DTH), and the effects of acute stressors on plasma corticosterone and leukocyte redistribution. The effects of several commonly used laboratory stressors including restraint, forced swim, isolation, and low ambient temperatures (4 degrees C) were examined. Exposure to each stressor elevated corticosterone concentrations, with restraint (a putative psychological stressor) evoking a significantly higher glucocorticoid response than other stressors. Chronic restraint and forced swim enhanced the DTH response compared to the handled, low temperature, or isolation conditions. Restraint, low temperature, and isolation significantly increased trafficking of lymphocytes and monocytes compared to forced swim or handling. Generally, acute restraint, low temperature, isolation, and handling increased trafficking of lymphocytes and monocytes. Considered together, our results suggest that the different stressors commonly used in psychoneuroimmunology research may not activate the physiological stress response to the same extent. The variation observed in the measured immune responses may reflect differential glucocorticoid activation, differential metabolic adjustments, or both processes in response to specific stressors.

6-MBOA affects testis size, but not delayed-type hypersensitivity, in white-footed mice (Peromyscus leucopus).

Many rodents use day length to time reproduction to occur when resources are abundant, but some species also use supplementary environmental cues. One supplementary cue is the plant-derived compound, 6-methoxy-2-benzoxazolinone (6-MBOA). Most rodents grow their gonads in response to 6-MBOA in their diets, but it is presently unknown whether they also use 6-MBOA to adjust other aspects of physiology, specifically their immune systems. 6-MBOA is structurally similar to melatonin, and seasonal changes in rodent immune activities are often mediated by melatonin. We therefore predicted that white-footed mice (Peromyscus leucopus), which breed seasonally and are reproductively sensitive to melatonin, would adjust their immune systems when fed 6-MBOA. 6-MBOA treated mice in long day lengths regressed their testes to a greater extent than mice fed a standard diet, or mice kept in short day lengths and fed 6-MBOA or a standard diet. One type of immune activity (delayed-type hypersensitivity) was not affected by 6-MBOA, however, although responses were greater in short versus long day mice. In sum, P. leucopus responded reproductively to 6-MBOA, although differently than other species; immune activity was unaffected. Other aspects of the immune system, especially in herbivorous rodents, may be affected by 6-MBOA and thus warrant further study.

Photoperiod affects estrogen receptor alpha, estrogen receptor beta and aggressive behavior.

Estrogens have important effects on male and female social behavior. Despite growing knowledge of the anatomy and behavioral effects of the two predominant estrogen receptor subtypes in mammals (ERalpha and ERbeta), relatively little is known about how these receptors respond to salient environmental stimuli. Many seasonally breeding species respond to changing photoperiods that predict seasonal changes in resource availability. We characterized the effects of photoperiod on aggressive behavior in two species of Peromyscus that exhibit gonadal regression in short days. P. polionotus (old field mice) were more aggressive than P. maniculatus (deer mice) and both species were more aggressive in short days. We used immunocytochemistry and real-time polymerase chain reaction to characterize the effects of photoperiod on ERalpha and ERbeta expression. In both species ERalpha-immunoreactive staining in the posterior bed nucleus of the stria terminalis (BNST) was increased in short vs. long days. Both species had reduced ERbeta-immunoreactive expression in the posterior BNST in short days. In the medial amygdala ERbeta immunoreactivity was increased in long days for both species. Using real-time polymerase chain reaction on punch samples that included the BNST, we observed that ERalpha mRNA was increased and ERbeta mRNA was decreased in short days. These data suggest that the effects of photoperiod on ERalpha and ERbeta expression may thus have important behavioral consequences.

Short days increase hypothalamic-pituitary-adrenal axis responsiveness.

Individuals dramatically alter physiology and behavior to adapt to seasonal changes in their environment. To cope with winter stressors such as reduced food availability and low temperatures, central stress responses are presumably modulated at the level of the hypothalamic-pituitary-adrenal (HPA) axis, but the details remain unspecified. We examined the effects of long or short photoperiods (day lengths) on corticosterone responses to restraint, HPA negative feedback sensitivity, glucocorticoid receptor gene expression in the hippocampus, the role of corticosterone in spatial learning, and corticosterone responses to stressors associated with the spatial water maze task in adult male white-footed mice (Peromyscus leucopus). Short days increased corticosterone responses to restraint, increased hippocampal glucocorticoid receptor expression, enhanced corticosterone negative feedback on the HPA axis, and increased sensitivity to dexamethasone suppression of corticosterone. Although spatial learning and memory performance (via water maze) of all mice was impaired after pharmacological corticosterone inhibition, both water maze exposure and treatment injections alone were sufficient to increase short-day, but not long-day, corticosterone concentrations. Thus, the effects of corticosterone on spatial learning in these mice may be complicated by photoperiodic differences in stressor response to the learning task itself. Overall, these results suggest that photoperiod-evoked modification of the HPA axis and its potential behavioral consequences may be adaptive for winter survival.

Behavioural alterations in male mice lacking the gene for D-aspartate oxidase.

D-serine and D-aspartate are important regulators of mammalian physiology. D-aspartate is found in nervous and endocrine tissue, specifically in hypothalamic supraoptic and paraventricular nuclei, pituitary, and adrenal medullary cells. Endogenous D-aspartate is selectively degraded by D-aspartate oxidase. We previously reported that adult male mice lacking the gene for D-aspartate oxidase (Ddo(-/-) mice) display elevated concentrations of D-aspartate in several neuronal and neuroendocrine tissues as well as impaired sexual performance and altered autogrooming behaviour. In the present study, we analyzed behaviours relevant to affect, cognition, and motor control in Ddo(-/-) mice. Ddo(-/-) mice display deficits in sensorimotor gating and motor coordination as well as reduced immobility in the forced swim test. Basal corticosterone concentrations are elevated. The Ddo(-/-) mice have D-aspartate immunoreactive cells in the cerebellum and adrenal glands that are not observed in the wild-type mice. However, no differences in anxiety-like behaviour are detected in open field or light-dark preference tests. Also, Ddo(-/-) mice do not differ from wild-type mice in either passive avoidance or spontaneous alternation tasks. Although many of these behavioural deficits may be due to the lack of Ddo during development, our results are consistent with the widespread distribution of D-aspartate and the hypothesis that endogenous D-aspartate serves diverse behavioural functions.

Social interactions alter proinflammatory cytokine gene expression and behavior following endotoxin administration.

Sick animals display a constellation of behaviors, including anhedonia, anorexia, and reduced social interactions. Acute infection eliminates female mating behavior, but fails to attenuate mating behavior in male rats. These results have been attributed to the different reproductive strategies and parental investment of the two sexes. Males putatively suppress the symptoms of infection in order to "deceive" females into mating. We sought to investigate the mechanisms responsible for this suppression. Adult male CD-1 mice were treated with lipopolysaccharide (LPS; a component of bacterial cell walls; 400 microg/kg), then paired 2 h later with a receptive female or juvenile male or remained isolated. Blood samples and brains of the males were collected 3 h post-LPS; hypothalamic interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNFalpha) gene expression was measured using RT-PCR. Contrary to our prediction, exposure to a female increased hypothalamic IL-1 and TNFalpha gene expression. LPS treatment significantly decreased testosterone and increased corticosterone secretions. Social interactions altered absolute corticosterone concentrations in saline-injected animals only. In order to determine whether increased production of hypothalamic cytokines reflected increased severity of sickness responses, body temperature was monitored in a second group of mice implanted with telemetric transmitters. Body mass, food intake, and consumption of sweetened condensed milk (a highly favored food) were also monitored in these mice for 72 h post-injection. LPS injections reduced milk intake, an effect that was modulated by social interactions; however, fever was unaltered relative to isolated animals. These results suggest that social interactions can adjust behavioral responses to infection although the ultimate cause of this adjustment remains unspecified.

Hypothalamic gene expression in reproductively photoresponsive and photorefractory Siberian hamsters.

An interval timing mechanism in the brain governs reproduction in seasonally breeding mammals by triggering refractoriness to inhibitory short photoperiods during midwinter. The neural mechanisms responsible for the timing and induction of photorefractoriness by this seasonal clock are unknown. Using cDNA microarrays and RT-PCR, we identified a class of genes encoding thyroxine (T4)-binding proteins (transthyretin, T4-binding globulin, albumin) whose expression is associated with reproductive refractoriness to short day lengths. Down-regulation of these genes was associated with reduced hypothalamic T4 uptake, which was reversed by long-day photoperiod treatments that restored responsiveness to short days. Circulating T4 concentrations did not vary with states of photoresponsiveness in euthyroid hamsters, but blockade of thyroid function accelerated the onset of photorefractoriness to short days. These data link changes in gene expression in the hypothalamus to the functional output of a seasonal clock. Reproductive inhibition in short days depends on T4 only late in the nonbreeding season. Down-regulation of genes encoding T4-binding proteins in the hypothalamus during this interval may restrict access of a static T4 signal to hypothalamic target tissues that regulate reproduction, thereby timing annual transitions in reproductive function. Hypothalamic autoregulation of T4 influx may constitute a critical cellular process involved in the generation and expression of seasonal reproductive rhythms and suggests a previously undescribed mechanism by which neural targets gain access to peripheral hormones.

Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels.

Golden-mantled ground squirrels (Spermophilus lateralis) undergo seasonal hibernation during which core body temperature (T(b)) values are maintained 1-2 degrees C above ambient temperature. Hibernation is not continuous. Squirrels arouse at approximately 7-day intervals, during which T(b) increases to 37 degrees C for approximately 16 h; thereafter, they return to hibernation and sustain low T(b)s until the next arousal. Over the course of the hibernation season, arousals consume 60-80% of a squirrel's winter energy budget, but their functional significance is unknown and disputed. Host-defense mechanisms appear to be downregulated during the hibernation season and preclude normal immune responses. These experiments assessed immune function during hibernation and subsequent periodic arousals. The acute-phase response to bacterial lipopolysaccharide (LPS) was arrested during hibernation and fully restored on arousal to normothermia. LPS injection (ip) resulted in a 1-1.5 degrees C fever in normothermic animals that was sustained for > 8 h. LPS was without effect in hibernating squirrels, neither inducing fever nor provoking arousal, but a fever did develop several days later, when squirrels next aroused from hibernation; the duration of this arousal was increased sixfold above baseline values. Intracerebroventricular infusions of prostaglandin E(2) provoked arousal from hibernation and induced fever, suggesting that neural signaling pathways that mediate febrile responses are functional during hibernation. Periodic arousals may activate a dormant immune system, which can then combat pathogens that may have been introduced immediately before or during hibernation.