Evaluation of the neutrophil:lymphocyte ratio as an indicator of chronic distress in the laboratory mouse.
ABSTRACT: When evaluating the effect of husbandry and biomethodologies on the well-being of laboratory mice, it is critical to utilize measurements that allow the distinguishing of acute stress from chronic stress. One of the most common measurements of stress in laboratory animals is the corticosterone assessment. However, while this measurement provides a highly accurate reflection of the animal's response to acute stressors, its interpretation is more prone to error when evaluating the effect of chronic stress. This study evaluated the use of the neutrophil:lymphocyte (NE:LY) ratio as an assessment of chronic stress in male and female C57Bl/6N mice as compared to serum corticosterone. One group of mice was exposed to mild daily stressors for 7 days, while the control group was handled with normal husbandry. The NE:LY ratio and serum corticosterone levels were significantly elevated in the chronically stressed mice, though a significant increase in corticosterone was only significant in males when compared by sex. The chronically stressed mice also demonstrated significantly fewer entries into the open arms and less time spent in the open arms of the elevated plus maze, suggesting that the mild daily stressors had induced a state of distress. The findings of this study confirm that the NE:LY ratio is a valid measurement for chronic stress in the laboratory mouse. However, these assays do not distinguish between distress or eustress, so behavioral and physiological assessments should always be included to determine a complete assessment of the well-being of the mouse.
Project description:BACKGROUND:Global biodiversity is decreasing at an alarming rate and amphibians are at the forefront of this crisis. Understanding the factors that negatively impact amphibian populations and effectively monitoring their health are fundamental to addressing this epidemic. Plasma glucocorticoids are often used to assess stress in amphibians and other vertebrates, but these hormones can be extremely dynamic and impractical to quantify in small organisms. Transcriptomic responses to stress hormones in amphibians have been largely limited to laboratory models, and there have been few studies on vertebrates that have evaluated the impact of multiple stressors on patterns of gene expression. Here we examined the gene expression patterns in tail tissues of stream-dwelling salamanders (Eurycea tynerensis) chronically exposed to the stress hormone corticosterone under different temperature regimes. RESULTS:We found unique transcriptional signatures for chronic corticosterone exposure that were independent of temperature variation. Several of the corticosterone responsive genes are known to be involved in immune system response (LY-6E), oxidative stress (GSTM2 and TRX), and tissue repair (A2M and FX). We also found many genes to be influenced by temperature (CIRBP, HSC71, HSP40, HSP90, HSP70, ZNF593). Furthermore, the expression patterns of some genes (GSTM2, LY-6E, UMOD, ZNF593, CIRBP, HSP90) show interactive effects of temperature and corticosterone exposure, compared to each treatment alone. Through a series of experiments we also showed that stressor induced patterns of expression were largely consistent across ages, life cycle modes, and tissue regeneration. CONCLUSIONS:Outside of thermal stressors, the application of transcriptomes to monitor the health of non-human vertebrate systems has been vastly underinvestigated. Our study suggests that transcriptomic patterns harbor stressor specific signatures that can be highly informative for monitoring the diverse stressors of amphibian populations.
Project description:Social stress (SS) has been linked to the development of cardiovascular disease (CVD), which is closely associated with insulin resistance (IR); however, the causal effect of SS on IR remains unclear. The 8-week-old male C57BL/6 mice were exposed to SS by housing with a larger CD-1 mouse in a shared home cage without physical contact for 10 consecutive days followed by high-fat diet (HFD) feeding. Control mice were housed in the same cage without a CD-1 mouse. After 6 weeks of HFD, insulin sensitivity was significantly impaired in stressed mice. While the percentage of classically activated macrophages in epididymal white adipose tissue (eWAT) was equivalent between the two groups, the percentage of lymphocyte antigen 6 complex locus G6D (Ly-6G)/neutrophil elastase (NE)-double positive cells markedly increased in stressed mice, accompanied by augmented NE activity assessed by ex vivo eWAT fluorescent imaging. Treatment with an NE inhibitor completely abrogated the insulin sensitivity impairment of stressed mice. In vitro NE release upon stimulation with a formyl peptide receptor 1 agonist was significantly higher in bone marrow neutrophils of stressed mice. Our findings show that SS-exposed mice are susceptible to the development of HFD-induced IR accompanied by augmented NE activity. Modulation of neutrophil function may represent a potential therapeutic target for SS-associated IR.
Project description:C1 neurons, located in the medulla oblongata, mediate adaptive autonomic responses to physical stressors (for example, hypotension, hemorrhage and presence of lipopolysaccharides). We describe here a powerful anti-inflammatory effect of restraint stress, mediated by C1 neurons: protection against renal ischemia-reperfusion injury. Restraint stress or optogenetic C1 neuron (C1) stimulation (10 min) protected mice from ischemia-reperfusion injury (IRI). The protection was reproduced by injecting splenic T cells that had been preincubated with noradrenaline or splenocytes harvested from stressed mice. Stress-induced IRI protection was absent in Chrna7 knockout (a7nAChR-/-) mice and greatly reduced by destroying or transiently inhibiting C1. The protection conferred by C1 stimulation was eliminated by splenectomy, ganglionic-blocker administration or ?2-adrenergic receptor blockade. Although C1 stimulation elevated plasma corticosterone and increased both vagal and sympathetic nerve activity, C1-mediated IRI protection persisted after subdiaphragmatic vagotomy or corticosterone receptor blockade. Overall, acute stress attenuated IRI by activating a cholinergic, predominantly sympathetic, anti-inflammatory pathway. C1s were necessary and sufficient to mediate this effect.
Project description:Exposure to chronic stress often elevates basal circulating glucocorticoids during the circadian nadir and leads to exaggerated glucocorticoid production following exposure to subsequent stressors. While glucocorticoid production is primarily mediated by the hypothalamic-pituitary-adrenal (HPA) axis, there is evidence that the sympathetic nervous system can affect diurnal glucocorticoid production by direct actions at the adrenal gland. Experiments here were designed to examine the role of the HPA and sympathetic nervous system in enhancing corticosterone production following chronic stress. Rats were exposed to a four-day stress paradigm or control conditions then exposed to acute restraint stress on the fifth day to examine corticosterone and ACTH responses. Repeated stressor exposure resulted in a small increase in corticosterone, but not ACTH, during the circadian nadir, and also resulted in exaggerated corticosterone production 5, 10, and 20min following restraint stress. While circulating ACTH levels increased after 5min of restraint, levels were not greater in chronic stress animals compared to controls until following 20min. Administration of astressin (a CRH antagonist) prior to restraint stress significantly reduced ACTH responses but did not prevent the sensitized corticosterone response in chronic stress animals. In contrast, administration of chlorisondamine (a ganglionic blocker) returned basal corticosterone levels in chronic stress animals to normal levels and reduced early corticosterone production following restraint (up to 10min) but did not block the exaggerated corticosterone response in chronic stress animals at 20min. These data indicate that increased sympathetic nervous system tone contributes to elevated basal and rapid glucocorticoid production following chronic stress, but HPA responses likely mediate peak corticosterone responses to stressors of longer duration.
Project description:Restraint and cold stress increase both corticosterone and glycemia, which lead to oxidative damages in hepatic tissue. This study assessed the effect of royal jelly (RJ) supplementation on the corticosterone level, glycemia, plasma enzymes and hepatic antioxidant system in restraint and cold stressed rats. Wistar rats were allocated into no-stress, stress, no-stress supplemented with RJ and stress supplemented with RJ groups. Initially, RJ (200mg/Kg) was administered for fourteen days and stressed groups were submitted to chronic stress from the seventh day. The results showed that RJ supplementation decreases corticosterone levels and improves glycemia control after stress induction. RJ supplementation also decreased the body weight, AST, ALP and GGT. Moreover, RJ improved total antioxidant capacity, SOD activity and reduced GSH, GR and lipoperoxidation in the liver. Thus, RJ supplementation reestablished the corticosterone levels and the hepatic antioxidant system in stressed rats, indicating an adaptogenic and hepatoprotective potential of RJ.
Project description:Many aspects of the laboratory environment are not tailored to the needs of rodents, which may cause stress. Unpredictable stressors can cause ulcers, prolonged pituitary-adrenal activation, and anhedonia. Similarly, pain has been demonstrated to slow wound healing, and mice experiencing pain exhibit altered behavior. However it is unknown how husbandry, which occurs when the mice are inactive, and lack of analgesia, specifically in a punch biopsy procedure, effects animal physiology, behavior, and welfare, particularly as it relates to sleep fragmentation. We hypothesized that sleep fragmentation, induced by unpredictable husbandry and lack of pain management will slow wound healing. Two main treatments were tested in a factorial design in C57BL/6 mice of both sexes (64 mice total); 1) analgesia (carprofen and saline) and 2) sleep disruptions (random and predictable). Mice were singly housed in a non-invasive sleep monitoring apparatus on arrival (Day -4). Disruption treatments were applied from Day -3 to 2. All mice received a punch biopsy surgery (Day 0) with topical lidocaine gel and their analgesic treatment prior to recovery, and on Days 1 and 2. Nesting behavior was assessed daily and a sugar cereal consumption test, as a measure of anhedonia, was conducted on Days -1 to 2. On Day 3, mice were euthanized and wound tissue and adrenal glands were collected. We found that the disruption predictability had no effect on mouse sleep, wound healing, or adrenal cortex:medulla ratio. It's possible that the disruption period was not long enough to induce chronic stress. However, male mice who received analgesia slept more than their female counterparts; this may be related to sex differences in pain perception. Overall, it does not appear that the predictability of disturbance effects sleep fragmentation or stress responses, indicating that husbandry activities do not need to occur at set predictable times to improve welfare.
Project description:Recent data suggest that, in animals living in social groups, stress-induced changes in behavior have the potential to act as a source of information, so that stressed individuals could themselves act as stressful stimuli for other individuals with whom they interact repeatedly. Such form of cross-over of stress may be beneficial if it enhances adaptive responses to ecological stressors in the shared environment. However, whether stress can be transferred among individuals during early life in natural populations remains unknown. Here we tested the effect of living with stressed siblings in a gull species where, as in many vertebrates, family represents the basic social unit during development. By experimentally modifying the level of stress hormones (corticosterone) in brood mates, we demonstrate that the social transfer of stress level triggers similar stress responses (corticosterone secretion) in brood bystanders. Corticosterone-implanted chicks and their siblings were faster in responding to a potential predator attack than control chicks. In gulls, fast and coordinated reactions to predators may increase the chances of survival of the whole brood, suggesting a beneficial fitness value of cross-over of stress. However, our data also indicate that living with stressed brood mates early in life entails some long-term costs. Near independence, fledglings that grew up with stressed siblings showed reduced body size, high levels of oxidative damage in lipids and proteins, and a fragile juvenile plumage. Overall, our results indicate that stress cross-over occurs in animal populations and may have important fitness consequences.
Project description:Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.
Project description:Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in anxiety-related brain structures. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In male rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. Here, we examined whether CVS would sensitize the behavioral and/or endocrine response to a subthreshold BNST PACAP infusion. Male and cycling female rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, rats were bilaterally infused into the BNST with a normally subthreshold dose of PACAP. We found an increase in startle amplitude and plasma corticosterone levels 30?min following intra-BNST PACAP infusion in male rats that had been previously exposed to CVS. CVS did not enhance the startle response in cycling females. Equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels even in previously stressed male rats. These results suggest that repeated exposure to stressors may differentially alter the neural circuits underlying the responses to intra-BNST PACAP, and may result in different anxiety-like responses in males and females.
Project description:Cycles of alcohol and stress are hypothesized to contribute to alcohol use disorders. How this occurs is poorly understood, although both alcohol and stress activate the neuroimmune system-the immune molecules and cells that interact with the nervous system. The effects of alcohol and stress on the neuroimmune system are mediated in part by peripheral signaling molecules. Alcohol and stress both enhance immunomodulatory molecules such as corticosterone and endotoxin to impact neuroimmune cells, such as microglia, and may subsequently impact neurons. In this study, we therefore examined the effects of acute and chronic ethanol (EtOH) on the corticosterone, endotoxin, and microglial and neuronal response to acute stress.Male Wistar rats were treated intragastrically with acute EtOH and acutely stressed with restraint/water immersion. Another group of rats was treated intragastrically with chronic intermittent EtOH and acutely stressed following prolonged abstinence. Plasma corticosterone and endotoxin were measured, and immunohistochemical stains for the microglial marker CD11b and neuronal activation marker c-Fos were performed.Acute EtOH and acute stress interacted to increase plasma endotoxin and microglial CD11b, but not plasma corticosterone or neuronal c-Fos. Chronic EtOH caused a lasting sensitization of stress-induced plasma endotoxin, but not plasma corticosterone. Chronic EtOH also caused a lasting sensitization of stress-induced microglial CD11b, but not neuronal c-Fos.These results find acute EtOH combined with acute stress enhanced plasma endotoxin, as well as microglial CD11b in many brain regions. Chronic EtOH followed by acute stress also increased plasma endotoxin and microglial CD11b, suggesting a lasting sensitization to acute stress. Overall, these data suggest alcohol and stress interact to increase plasma endotoxin, resulting in enhanced microglial activation that could contribute to disease progression.