Stressed Mouse Research Articles

Changes of peripheral immunity in adult mice with early life stress

Objective To investigate the effect of early life stress on peripheral immunity in adult in mice. Methods The neonatal mice were maternal seperation 2 hours everyday from day 2~(nd) to 21~(st), and breeding alone after day 22~(nd). The differential white blood cell count were conducted with blood smear. Concanavalin (Con A) and lipopolysaccharide( LPS) induced splentic proliferation were measured with MTT method, and the effect of serum from stressed mouse on normal lymphocte proliferation were also tested with MTT. Results Adult mice with early life stress showed that neutrophile granulocytes increased from (17. 94 ±1.24)%,( 17. 33 ± 1.80)% and (21.25 ±2.82)% to (27.17 ±3.19)% ,(29.75 ±2.76)% and (28.33 ±3.12)% at 1,2 and 3 months old. Lymphocytes decreased from (79. 06 ± 1. 18 )%, (77. 83 ± 3. 05 )% and (72. 46 ± 4. 45 )% to (69.72 ±4.97)% ,(64.00 ±3.9)% and (66.74 ±2.67)% at 1,2 and 3 months old,and monocyte increased as well. Both Con A and LPS induced splentic proliferation increased in mice with early life stress at 1,2 and 3 months old. Sera from mice with early life stress decreased the normal lymphocyte proliferation compared with sera from control mice. Conclusion Early life maternal deprivation increase cellular immunity in adulthood in mice. Key words: Early life stress; Maternal deprivation; Adult; Immunity function

Mutant mouse models of oxidative stress

Oxidative stress corresponds to an excess in reactive oxygen species (ROS) including free radicals which are highly reactive with cellular constituents. Thereby ROS induce damage to DNA, proteins and lipids, which are all involved in the etiology of numerous pathologies such as cancer. To prevent potential damage, a tight regulation of ROS level is achieved through numerous enzyme systems and small molecules such as glutathione and vitamin C. Mutant mouse models targeting antioxidant enzymes have confirmed their essential role in ROS level control, and have shown a limited redundancy of their activity. Additionally, a number of other mutant mouse models exhibit increased ROS levels, suggesting an antioxidant role for the corresponding targeted gene. This is the case for mice deficient for the transcription factors p53, JunD, FoxOs, and HIF-2alpha, which are involved in the modulation of antioxidant enzymes expression. Mice deficient either for the stress factor TP53INP1, which is a target of p53, or for ATM involved in DNA damage sensoring, also show a constitutive oxidative stress. Finally, the last reported case of mice with a permanent oxidative stress targets Bmi which is a transcriptional repressor of the polycomb family. Interestingly, most of these "oxidative stressed mice" either spontaneously develop cancers or are more susceptible than wild-type to tumor-induced protocols. Altogether, these models markedly reinforce the causal link between oxidative stress and cancer. In the future, they will be helpful tools for basic research aimed at unraveling the interplay between redox control actors as well as their relative importance. In addition, these oxidative stressed mouse models may be useful for applied research in particular in preclinical assays where redox status regulation is absolutely required.

Post-stress chemosignals affect cells from immunocompetent organs in laboratory mice of three inbred strains

Quantity of antibody producing cells and changes in bone marrow dividing cells of mouse males were studied after the exposure with chemosignals from intact or stressed donor mouse males. Inbred CBA, BALB/c and C57BL/6 strains were used. It is shown that excreted volatiles decrease quantity of antibody producing cells in spleen and at the same time raise the level of mitotic disturbances in bone marrow cells of recipient mice. Pheromone effect depends on genotype and physiological state of the recipients. For the first time we describe here the influence of mouse female pheromone 2,5-dimethylpyrazine on analyzed features. Biological meaning of the discovered effects is discussed.

The antistress effect of ginseng total saponin and ginsenoside Rg3 and Rb1 evaluated by brain polyamine level under immobilization stress

The present study aims to demonstrate the ability of ginseng total saponin (GTS), ginsenosides Rg3 and Rb1 to reduce brain polyamine levels in immobilization-stressed gerbil mice. A previous study reported that ginsenosides had an anti-stress property. So, we tested the anti-stress effect of ginseng by investigating the brain level of polyamine, a well-known stress stimuli marker. We determined the brain polyamine levels under 30-min immobilization stress in pretreating GTS (100 mg kg −1, oral), ginsenosides Rg3 and Rb1 (10 mg kg −1, oral, respectively). Then, we compared polyamine levels between the non-stressed mouse and the stressed mouse which had taken saline orally to check the placebo effect. Putrescine (PUT) levels were significantly increased ( P < 0.01) in the stressed condition, but it was reduced in pretreatment of GTS, ginsenosides Rg3 ( P < 0.01, respectively) and Rb1 ( P < 0.001) under 30-min immobilization stressed-mouse. However, other polyamine levels did not change regardless of stressed condition or GTS-, ginsenosides Rg3- and Rb1-treated stressed condition. These results mean that only PUT could be a marker for stress and GTS, ginsenosides Rg3 and Rb1 administration lead to an anti-stress effect. Thus, our studies indicate that GTS, ginsenosides Rg3 and Rb1 may play a neuroprotective role in the immobilization-stressed brain.

Acetylation of Human 8-Oxoguanine-DNA Glycosylase by p300 and Its Role in 8-Oxoguanine Repair In Vivo

The human 8-oxoguanine-DNA glycosylase 1 (OGG1) is the major DNA glycosylase responsible for repair of 7,8-dihydro-8-oxoguanine (8-oxoG) and ring-opened fapyguanine, critical mutagenic DNA lesions that are induced by reactive oxygen species. Here we show that OGG1 is acetylated by p300 in vivo predominantly at Lys338/Lys341. About 20% of OGG1 is present in acetylated form in HeLa cells. Acetylation significantly increases OGG1's activity in vitro in the presence of AP-endonuclease by reducing its affinity for the abasic (AP) site product. The enhanced rate of repair of 8-oxoG in the genome by wild-type OGG1 but not the K338R/K341R mutant, ectopically expressed in oxidatively stressed OGG1-null mouse embryonic fibroblasts, suggests that acetylation increases OGG1 activity in vivo. At the same time, acetylation of OGG1 was increased by about 2.5-fold after oxidative stress with no change at the polypeptide level. OGG1 interacts with class I histone deacetylases, which may be responsible for its deacetylation. Based on these results, we propose a novel regulatory function of OGG1 acetylation in repair of its substrates in oxidatively stressed cells.

Cardiac Myosin-Binding Protein-C Phosphorylation and Cardiac Function

The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C(AllP-)). A TG line showing &40% replacement with MyBP-C(AllP-) showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C(AllP-), the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C(AllP-) was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C(t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C(t/t) animals. In contrast, when the MyBP-C(t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-CWT), the MyBP-C(t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function.

Acquisition of Unprecedented Phosphatidylinositol 3,5-Bisphosphate Rise in Hyperosmotically Stressed 3T3-L1 Adipocytes, Mediated by ArPIKfyve-PIKfyve Pathway

Unlike yeast, where hyperosmotic stress induces a dramatic increase in phosphatidylinositol 3,5-bisphosphate (PtdIns 3,5-P(2)) synthesis, in mammalian cells, although activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P(2), indicating the PtdIns 3,5-P(2) pathway is not involved in mammalian osmo-protective responses. Here we report an unexpected and marked PtdIns 3,5-P(2) increase in response to hyperosmotic stress in differentiated 3T3-L1 adipocytes. Because this effect was not observed in the precursor preadipocytes, a specific role during acquisition of the adipocyte phenotype and transition into insulin-responsive cells could be suggested. However, acute insulin action did not result in a measurable PtdIns 3,5-P(2) rise, indicating the PtdIns 3,5-P(2) pathway is a specific hyperosmotically activated signaling cascade selectively operating in differentiated 3T3-L1 adipocytes. Hyperosmolarity activates different components of several kinase cascades, including p38 mitogen-activated protein and tyrosine kinases, but these appear to be separate from the activated PtdIns 3,5-P(2) pathway. Because PtdIns 3,5-P(2) is primarily produced by PIKfyve-catalyzed synthesis and requires the upstream activator hVac14 (called herein ArPIKfyve) that physically associates with and activates PIKfyve, we examined the contribution of ArPIKfyve-PIKfyve for the hyperosmotic stress-induced rise in PtdIns 3,5-P(2). Small interfering RNA-directed gene silencing to selectively deplete ArPIKfyve or PIKfyve in 3T3-L1 adipocytes determined the ArPIKfyve-PIKfyve axis fully accountable for the hyperosmotically activated PtdIns 3,5-P(2). Together these results reveal a previously uncharacterized PtdIns 3,5-P(2) pathway activated selectively in hyperosmotically stressed 3T3-L1 adipocytes and suggest a plausible role for PtdIns 3,5-P(2) in the osmo-protective response mechanism in this cell type.

Determination of corticosterone in mouse plasma by a sweeping technique using micellar electrokinetic chromatography

The separation and on-line concentration of corticosterone in mouse blood was achieved by means of capillary electrophoresis/UV absorbance using sodium dodecyl sulfate (SDS) as a surfactant. The procedure involved the use of an on-line sample concentration method by sweeping-micellar electrokinetic chromatography (sweeping-MEKC). Optimal on-line concentration and separation conditions were determined. The detection limit for this method was 5 ng/ml ( S/ N=3) and photodiode array detection at 247 nm was used for identification. For the analysis of actual samples, corticosterones from blood samples of a non-stressed and stressed mouse were determined. The results show that only a minor amount of corticosterone was produced by a non-stressed mouse, whereas a significant amount was present in the blood sample from a stressed mouse. The method developed here can be used to examine corticosterone levels as a marker of stress in test animals and may also be used for estimating the effect of stress-release medications.

Melatonin reduces interleukin secretion in amyloid-β stressed mouse brain slices

Neurodegeneration in Alzheimer's disease (AD) is associated with many features of the immune system. For example, cytokines such as IL-6, synthesized by microglia and astrocytes, are associated with senile plaques. To further study the role of cytokines in early stage AD neurodegeneration, an organotypic mouse brain slice culture system with microglia and astrocytes was developed. Amyloid-β(Aβ1–40) induced the secretion of both IL-1β and IL-6. Melatonin, an antioxidant and pineal hormone, reduced interleukin secretion in a concentration dependent manner. We also observed that melatonin, alone, had no effect on IL-1β secretion but at a concentration of 500 μM induced the secretion of IL-6. This organotypic slice system can be used to study the early role of immune system molecules on neurodegeneration.

Metallothionein Induction in Response to Restraint Stress: TRANSCRIPTIONAL CONTROL, ADAPTATION TO STRESS, AND ROLE OF GLUCOCORTICOID

Metallothioneins (MT) have been implicated in the protection of cells from oxidative stress. We studied the molecular mechanism of induction of MT-I and MT-II in response to restraint stress using a mouse model system in which the animals were restrained in well ventilated polypropylene tubes for 12 h each day (one cycle). Here, we show that MT-I and MT-II mRNA levels were elevated as much as 10-20-fold after just one cycle of this simple stress. Stress-mediated MT induction occurred at the transcriptional level. The level of MT mRNA correlated with the stress-induced increase, and not with the diurnal variation, in the level of serum glucocorticoid. Treatment of the mice with RU 486, a glucocorticoid receptor antagonist, prior to restraint stress inhibited MT induction by at least 50%. Furthermore, the glucocorticoid responsive element-binding activity in the liver nuclear extracts from the stressed mice was significantly higher than that in the control mice. The complex formations between the transcription factor Sp1, MTF1, or MLTF/ARE and the respective specific oligonucleotides were not altered in the liver from the stressed mouse. The MT mRNA levels returned to the basal level at the end of nine cycles of stress, indicating habituation of the animals to restraint stress. At this stage, exposure of the animals to another type of stress, treatment with heavy metals, resulted in further induction of MT. These data indicate that glucocorticoid is the primary physiological factor responsible for MT induction following restraint stress, and the glucocorticoid receptor is the major transcription factor involved in this process.

Identification of novel stress-induced genes downstream of chop.

CHOP (GADD153) is a small nuclear protein that dimerizes avidly with members of the C/EBP family of transcription factors. Normally undetectable, it is expressed at high levels in cells exposed to conditions that perturb protein folding in the endoplasmic reticulum and induce an endoplasmic reticulum stress response. CHOP expression in stressed cells is linked to the development of programmed cell death and, in some instances, cellular regeneration. In this study, representational difference analysis was used to compare the complement of genes expressed in stressed wild-type mouse embryonic fibroblasts with those expressed in cells nullizygous for chop. CHOP expression, in concert with a second signal, was found to be absolutely required for the activation by stress of a set of previously undescribed genes referred to as DOCs (for downstream of CHOP). DOC4 is a mammalian ortholog of a Drosophila gene, Tenm/Odz, implicated in patterning of the early fly embryo, whereas DOC6 encodes a newly recognized homolog of the actin-binding proteins villin and gelsolin. These results reveal the existence of a novel CHOP-dependent signaling pathway, distinct from the known endoplasmic reticulum unfolded protein response, which may mediate changes in cell phenotype in response to stress.

Experimental partial sympathicotonia, and effects of some drugs on it in restraint and water immersion stressed animals.

The contractile response to acetylcholine (ACh) of the isolated duodenum from the restraint and water immersion stressed (RWIS) mouse was found to be enhanced by the stress for 1 hr and reach a maximum in 3 hr stress followed by a decrease. It was clarified that this rise in the response is not due to the change in the affinity to ACh but due to the increase of the intrinsic activity. The contractile response to KC1 was augmented only when concentration of KC1 was high, While the response to BaCl2 was little enhanced in the stressed animal. The relaxing response to noradrenaline (NA) of the isolated duodenum from rat, on the other hand, was reduced by the stress. Such reduced response to NA was observed to be more marked than the enhanced response to ACh in the vas deferens isolated from the stressed animal. The pretreatment to the RWIS mouse with either antiadrenergic or cholinergic drugs resulted in the clear-cut blockade of the enhancement of response to ACh of the isolated duodenum from this animal. These results contrasted to the effects on the reduced response to ACh of the isolated duodenum from the SART stressed (repeated cold stressed) mouse. The pre-administration of psychotropic drugs showed marked suppression on the enhancement of response of the duodenum of the RWIS animal, though there was a quantitative difference between the RWIS and SART animals. The pretreatment with a neurosedative, Neurotropin (NSP) was also found to show similarly marked suppressive action. From these results, it was considered that the duodenum and the vas deferens of the RWIS animal are in the state of the sympathicotonia, namely partial sympathicotonia.