Response In Female Mice Research Articles

Protective role of Nrf2 in zinc oxide nanoparticles-induced lung inflammation in female mice and sexual dimorphism in susceptibility

BackgroundZinc oxide nanoparticles (ZnO-NPs) are currently employed in various products such as rubber, paint, and cosmetics. Our group reported recently that Nrf2 protein provides protection against pulmonary inflammation induced by ZnO-NPs in male mice. The current study investigated the effect of Nrf2 deletion on the lung inflammatory response in female mice exposed to ZnO-NPs. MethodsAn equal number of female Nrf2-/- mice and female Nrf2+/+ mice (24 each) were allocated into three equal groups, and each was exposed to ZnO-NPs at either 0, 10 or 30 µg ZnO-NPs/mouse through pharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were examined 14 days later to determine the number of inflammatory cells, the protein level, and for scoring inflammation histopathologically. The mRNA levels of Nrf2-dependent antioxidant enzymes and proinflammatory cytokine in lung tissue were also measured. ResultsExposure to ZnO-NPs increased all types of BALF cells and lung inflammation scores in both of female Nrf2-null (Nrf2-/-) and wild-type (Nrf2+/+) mice, and Nrf2 deletion enhanced ZnO-NPs-induced increase in the number of eosinophils in BALF. Exposure to ZnO-NPs dose-dependently increased the level of oxidized glutathione (GSSG), and mRNA levels of proinflammatory cytokines/chemokines; KC, MIP-2, IL-6, IL-1β and MCP-1 only in wild-type mice. Nrf2 deletion decreased total glutathione levels and basal mRNA levels of SOD1 and NQO1, and increased the basal mRNA level of above proinflammatory cytokines/chemokines. Nrf2 deletion enhanced ZnO-NPs-induced downregulation of GcLc, GR and TGF-β and upregulation of HO-1 and TNF-α. Taken together with our previous results in male mice, our results showed a lower susceptibility of females to lung tissue inflammation, relative to males, irrespective of Nrf2 deletion, and that enhancement of ZnO-NPs-induced upregulation of HO-1 and TNF-α and downregulation of GcLc, GR and TGF-β by deletion of Nrf2 is specific to female mice. ConclusionWe conclude that Nrf2 provides protection in female mice against increase in BALF eosinophils, probably through down-regulation of proinflammatory cytokines/chemokines and upregulation of oxidative stress-related genes. The study also suggests lower susceptibility to lung tissue inflammation in female mice relative to their male counterparts and the synergistic effects of Nrf2 and exposure to ZnO-NPs on mRNA expression of GcLc, GR, HO-1, TGF-β or TNF-α in female mice.

Comparison of inflammatory and behavioral responses to chronic stress in female and male mice

Major depressive disorder (MDD) is a debilitating disease with a high worldwide prevalence. Despite its greater prevalence in women, male animals are used in most preclinical studies of depression even though there are many sex differences in key components of depression, such as stress responses and immune system functions. In the present study, we found that chronic restraint stress-induced depressive-like behaviors are quite similar in male and female mice, with both sexes displaying increased immobility time in the tail suspension test and reduced social interactions, and both sexes exhibited deficits in working and spatial memories. However, in contrast to the similar depressive-like behaviors developed by male and female mice in response to stress, they displayed different patterns of pro-inflammatory cytokine increases in the periphery and the brain, different changes in microglia, and different changes in the expression of Toll-like receptor 4 in response to stress. Treatment with (+)-naloxone, a Toll-like receptor 4 antagonist that previously demonstrated anti-depressant-like effects in male mice, was more efficacious in male than female mice in reducing the deleterious effects of stress, and its effects were not microbiome-mediated. Altogether, these results suggest differential mechanisms to consider in potential sex-specific treatments of depression.

Deficiency of Ataxia Telangiectasia‐mutated Kinase (ATM) Preserves Heart Function by Affecting Cardiac Remodeling in Response to Western‐type Diet in Female Mice

BACKGROUNDWestern‐type Diet (WD) and deficiency of ATM protein independently associate with heart disease. Previous work demonstrated that WD in male ATM deficient mice induces accelerated body weight gain and heart dysfunction (Am J Physiol Heart Circ Physiol. 2021;320:H2324‐H2338). Conversely, WD in female ATM deficient mice attenuates weight gain and preserves heart function (unpublished data). Here, we investigated the mechanism by which ATM deficiency preserves heart function in female mice. It is hypothesized that ATM deficiency attenuates adverse myocardial remodeling in female mice, thereby preserving heart function.METHODSFemale wild‐type (WT) and ATM heterozygous knockout (hKO) mice, aged 6 weeks, were fed with normal chow (NC) or WD for 14 weeks. Heart sections were stained with Masson’s trichrome to quantify fibrosis, TUNEL‐stained to quantify apoptosis, and WGA (wheat germ agglutinin)‐stained to quantify myocyte hypertrophy. Data were analyzed using ANOVA followed by Student‐Newman‐Keuls test.RESULTSATM deficiency associated with increased fibrosis, apoptosis (myocytes and non‐myocytes) and hypertrophy in hKO‐NC vs WT‐NC (p<0.05; n=4). WD significantly increased fibrosis in WT‐WD mice, while no increase in fibrosis was observed in hKO‐WD. WD significantly increased apoptosis in both genotypes. However, the increase in apoptosis was significantly lower in hKO‐WD vs WT‐WD (p<0.05; n=4). WD increased hypertrophy in WT group. However, the hypertrophy remained significantly higher in hKO‐WD vs WT‐WD (p<0.05; n=4).CONCLUSIONDecrease in myocardial fibrosis and apoptosis, and increase in myocyte hypertrophy may play a role in preservation of heart function in ATM deficient female mice in response to WD.

Vascular and blood-brain barrier-related changes underlie stress responses and resilience in female mice and depression in human tissue

Prevalence, symptoms, and treatment of depression suggest that major depressive disorders (MDD) present sex differences. Social stress-induced neurovascular pathology is associated with depressive symptoms in male mice; however, this association is unclear in females. Here, we report that chronic social and subchronic variable stress promotes blood-brain barrier (BBB) alterations in mood-related brain regions of female mice. Targeted disruption of the BBB in the female prefrontal cortex (PFC) induces anxiety- and depression-like behaviours. By comparing the endothelium cell-specific transcriptomic profiling of the mouse male and female PFC, we identify several pathways and genes involved in maladaptive stress responses and resilience to stress. Furthermore, we confirm that the BBB in the PFC of stressed female mice is leaky. Then, we identify circulating vascular biomarkers of chronic stress, such as soluble E-selectin. Similar changes in circulating soluble E-selectin, BBB gene expression and morphology can be found in blood serum and postmortem brain samples from women diagnosed with MDD. Altogether, we propose that BBB dysfunction plays an important role in modulating stress responses in female mice and possibly MDD.

Prolactin signaling modulates stress-induced behavioral responses in a preclinical mouse model of migraine.

The aim of this study was to determine if prolactin signaling modulates stress-induced behavioral responses in a preclinical migraine model. Migraine is one of the most complex and prevalent disorders. The involvement of sex-selective hormones in migraine pathology is highly likely as migraine is more common in women and its frequency correlates with reproductive stages. Prolactin has been shown to be a worsening factor for migraine. Normally prolactin levels are low; however levels can surge during stress. Dopamine receptor agonists, which suppress pituitary prolactin release, are an effective migraine treatment in a subset of patients. Previously, we showed that administration of prolactin onto the dura mater induces female-specific behavioral responses, suggesting that prolactin may play a sex-specific role in migraine. The effects of prolactin signaling were assessed using a preclinical migraine model we published recently in which behavioral sensitization is induced by repeated stress. Plasma prolactin levels were assessed in naïve and stressed CD-1 mice (n=3-5/group) and transgenic mice with conditional deletion of the Prlr in Nav1.8-positive sensory neurons (Prlr conditional knock-out [CKO]; n=3/group). To assess the contribution of prolactin release during stress, naïve or stressed male and female CD-1 mice were treated with the prolactin release inhibitor bromocriptine (2mg/kg; n=7-12/group) or vehicle for 5days (8-12/group) and tested for facial hypersensitivity following stress. Additionally, the contribution of ovarian hormones in regulating the prolactin-induced responses was assessed in ovariectomized female CD-1 mice (n=6-10/group). Furthermore, the contribution of Prlr activation on Nav1.8-positive sensory neurons was assessed. Naïve or stressed male and female Prlr CKO mice and their control littermates were tested for facial hypersensitivity (n=8-9/group). Immunohistochemistry was used to confirm loss of Prlr in Nav1.8-positive neurons in Prlr CKO mice. The total sample size is n=245; the full analysis sample size is n=221. Stress significantly increased prolactin levels in vehicle-treated female mice (39.70±2.77; p<0.0001). Bromocriptine significantly reduced serum prolactin levels in stressed female mice compared to vehicle-treated mice (-44.85±3.1; p<0.0001). Additionally, no difference was detected between female stressed mice that received bromocriptine compared to naïve mice treated with bromocriptine (-0.70±2.9; p=0.995). Stress also significantly increased serum prolactin levels in male mice, although to a much smaller extent than in females (0.61±0.08; p<0.001). Bromocriptine significantly reduced serum prolactin levels in stressed males compared to those treated with vehicle (-0.49±0.08; p=0.002). Furthermore, bromocriptine attenuated stress-induced behavioral responses in female mice compared to those treated with vehicle (maximum effect observed on day 4 post stress [0.21±0.08; p=0.03]). Bromocriptine did not attenuate stress-induced behavior in males at any timepoint compared to those treated with vehicle. Moreover, loss of ovarian hormones did not affect the ability of bromocriptine to attenuate stress responses compared to vehicle-treated ovariectomy mice that were stressed (maximum effect observed on day 4 post stress [0.29±0.078; p=0.013]). Similar to CD-1 mice, stress increased serum prolactin levels in both Prlr CKO female mice (27.74±9.96; p=0.047) and control littermates (28.68±9.9; p=0.041) compared to their naïve counterparts. There was no significant increase in serum prolactin levels detected in male Prlr CKO mice or control littermates. Finally, conditional deletion of Prlr from Nav1.8-positive sensory neurons led to a female-specific attenuation of stress-induced behavioral responses (maximum effect observed on day 7 post stress [0.32±0.08; p=0.007]) compared to control littermates. These data demonstrate that prolactin plays a female-specific role in stress-induced behavioral responses in this preclinical migraine model through activation of Prlr on sensory neurons. They also support a role for prolactin in migraine mechanisms in females and suggest that modulation of prolactin signaling may be an effective therapeutic strategy in some cases.

Automated detection of squint as a sensitive assay of sex-dependent calcitonin gene-related peptide and amylin-induced pain in mice.

We developed an automated squint assay using both black C57BL/6J and white CD1 mice to measure the interpalpebral fissure area between the upper and lower eyelids as an objective quantification of pain. The automated software detected a squint response to the commonly used nociceptive stimulus formalin in C57BL/6J mice. After this validation, we used the automated assay to detect a dose-dependent squint response to a migraine trigger, the neuropeptide calcitonin gene-related peptide, including a response in female mice at a dose below detection by the manual grimace scale. Finally, we found that the calcitonin gene-related peptide amylin induced squinting behavior in female mice, but not males. These data demonstrate that an automated squint assay can be used as an objective, real-time, continuous-scale measure of pain that provides higher precision and real-time analysis compared with manual grimace assessments.

Bridging Sex-Specific Differences in the CAR-Mediated Hepatocarcinogenesis of Nitrapyrin Using Molecular and Apical Endpoints.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10/CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed.

An Exaggerated Immune Response in Female BALB/c Mice Controls Initial Toxoplasma gondii Multiplication but Increases Mortality and Morbidity Relative to Male Mice

Studies indicate that female mice are more susceptible to T. gondii infection, as defined by higher mortality rates in comparison to male mice. However, whether this is due to an inability to control initial parasite multiplication or due to detrimental effects of the immune system has not been determined. Therefore, the following studies were undertaken to determine the influence of sex on early parasite multiplication and the immune response during T. gondii infection and to correlate this with disease outcome. Early parasite replication was studied through applying an in vivo imaging system (IVIS) with luciferase expressing T. gondii. In parallel immunological events were studied by cytometric bead array to quantify key immunological mediators. The results confirmed the previous findings that female mice are more susceptible to acute infection, as determined by higher mortality rates and weight loss compared with males. However, conflicting with expectations, female mice had lower parasite burdens during the acute infection than male mice. Female mice also exhibited significantly increased production of Monocyte Chemoattractant Protein-1 (MCP-1), Interferon (IFN)-γ, and Tumour Necrosis Factor (TNF)-α than male mice. MCP-1 was found to be induced by T. gondii in a dose dependent manner suggesting that the observed increased levels detected in female mice was due to a host-mediated sex difference rather than due to parasite load. However, MCP-1 was not affected by physiological concentration of estrogen or testosterone, indicating that MCP-1 differences observed between the sexes in vivo are due to an as yet unidentified intermediary factor that in turn influences MCP-1 levels. These results suggest that a stronger immune response in female mice compared with male mice enhances their ability to control parasite replication but increases their morbidity and mortality.

17‐β‐Estradiol Causes a Moderate Increase in Blood Pressure in Kidney‐Specific BMAL1 Knockout and Control Mice in Response to Potassium Depleted, High Salt Diet

Blood pressure (BP) regulation is influenced by circadian clock factors. Previously, we reported a sex-specific BP response in kidney-specific BMAL1 knockout mice (KS-BMAL1 KO) where male KS-BMAL1 KO exhibited lower BP than control mice but females lacked this difference. BMAL1 is a core mammalian circadian transcription factor responsible for the tissue-specific regulation of thousands of genes. The goal of this study was to determine if female sex hormones, specifically 17-β-estradiol, contributes to the lack of BP response in female mice. We hypothesize that ovariectomy (OVX) without estradiol supplementation will replicate the phenotype found in male mice and reveal a genotype effect in females. OVX and subcutaneous pellet implantation was performed using either placebo or estradiol pellets (0.1 mg/60 day release) in adult female KS-BMAL1 KO and control mice. After recovery, telemetry devices were implanted. Mice were administered a potassium depleted, high salt diet (0KHS) regimen (0% K, 2% Na, N=3-4). Telemetry recordings of BP demonstrated mean arterial pressure was approximately 5±1 mmHg higher in estradiol treated mice compared to placebo (P=0.02). There was a trend that suggests estradiol exacerbates the BP response following 0KHS (P=0.05). There were no genotype differences. Estradiol supplementation increased heart rate in both genotypes by 11±4% (P=0.0001). Heart rate decreased by 21±2% in all groups following 0KHS treatment (P<0.0001). Differences in locomotor activity were not detected between any groups. Because there were no reported genotype differences, KS-BMAL1 KO and control mice data were combined to determine the effect of hormone replacement therapy on BP over the course of the whole treatment period. Two-way repeated measures ANOVA showed that estradiol supplementation increased BP in response to 0KHS (Interaction P=0.0002; Figure). In conclusion, our results indicate that BMAL1 does not play a role in estradiol regulated BP control contrary to our hypothesis. Surprisingly, estradiol supplementation leads to higher BP in a potassium depleted, high salt environment. These differences in BP may be related to estradiol-induced changes in heart rate. These findings provide insight on the previously reported sex-specific response to KS-BMAL1 KO in BP control. Importantly, the effect of estradiol supplementation on BP in this model of salt-sensitive hypertension must be further investigated due to the possible implications for post-menopausal BP regulation.

Early Chronic Methamphetamine Exposure Induces Cognitive Impairments and Oxidative Damage in Adult Mice

Prescription stimulant misuse among young adults has gained popularity in the United States over the last decade, with amphetamine compounds becoming the second most common illicit drug used in college students. Misuse of these drugs can pose serious risks due to their high potential for abuse that can lead to dependency and neurotoxicity. Furthermore, there are prominent sex differences in psychostimulant use: men use more frequently and women have higher success rates in treatment but are more vulnerable to dependence and adverse effects. Psychostimulants, even at moderate doses, have been shown to promote oxidative stress, via auto-oxidation or metabolism of accumulated redox active dopamine. Oxidative stress has also been implicated in neurobehavioral impairments. Therefore, exposure to psychostimulants as a consequence of abuse would provide a second hit predicted to increase brain impairments. Moreover, the mesocorticolimbic pathway, a critical dopaminergic pathway, is particularly vulnerable to oxidative insults, and reveals sex-dependent differences in dopamine dynamics. We hypothesized that oxidative stress may act as a prominent cellular mechanism in mediating psychostimulant-induced neurotoxicity and neurobehavioral deficits. The purpose of this pilot study was to determine the short-term consequences of chronic, low toxic dosing of the prototypical psychostimulant, methamphetamine (METH), on cognition and markers of oxidative stress and dopaminergic function. Groups of young male and female (4-month-old) C57BL/6J mice received non-contingent intraperitoneal injections of either saline or methamphetamine (1.4 mg/kg) twice a day for 4 weeks. Two weeks following the last injection, animals were tested for cognitive function using 3 different tests: Morris water maze, active avoidance, and fear conditioning. After the completion of behavioral testing, brain regions were dissected and markers of oxidative stress and dopaminergic function were assayed. METH administration appeared to induce deficits in spatial learning and memory in both males and females, and in active avoidance and fear conditioning response in female mice only. Markers of dopaminergic function do not seem to be majorly affected by METH administration, while markers of lipid peroxidation were increased in the cortex and hippocampus of METH-treated females. These preliminary results suggest that chronic administration of METH induced impairments in cognitive function and changes in oxidative stress. These data indicate functional and biochemical changes that may have long-term consequences on brain function which will be the focus of future studies.

Evaluation of Sex Differences in Murine Diabetic Ketoacidosis and Neutropenic Models of Invasive Mucormycosis.

There is increased concern that the quality, generalizability and reproducibility of biomedical research can be influenced by the sex of animals used. We studied the differences between male and female mice in response to invasive pulmonary mucormycosis including susceptibility to infection, host immune reaction and responses to antifungal therapy. We used diabetic ketoacidotic (DKA) or neutropenic mice infected with either Rhizopus delemar or Mucor circinelloides. The only difference detected was that when DKA mice were infected with M. circinelloides, female mice were more resistant to infection than male mice (median survival time of 5 vs. 2 days for female and male mice, respectively). However, a 100% lethality was detected among infected animals of both sexes. Treatment with either liposomal amphotericin B (L-AMB) or posaconazole (POSA) protected mice from infection and eliminated the difference seen between infected but untreated female and male mice. Treatment with L-AMB consistently outperformed POSA in prolonging survival and reducing tissue fungal burden of DKA and neutropenic mice infected with R. delemar or M. circinelloides, in both mouse sexes. While little difference was detected in cytokine levels among both sexes, mucormycosis infection in the DKA mouse model induced more inflammatory cytokines/chemokines involved in neutrophil (CXCL1) and macrophage (CXCL2) recruitment vs. uninfected mice. As expected, this inflammatory response was reduced in the neutropenic mouse model. Our studies show that there are few differences between female and male DKA or neutropenic mice infected with mucormycosis with no effect on the outcome of treatment or host immune response.

Assessment of Tumor Response in Mice with Ovarian Peritoneal Carcinomatosis using Doppler Ultrasound of the Superior Mesenteric Artery and Celiac Trunk

The goal of the work described here was to assess the performance of Doppler ultrasound (US) of the superior mesenteric artery (SMA) and celiac trunk (CT) in the evaluation of tumor response in female mice with ovarian peritoneal carcinomatosis treated either with bevacizumab or with carboplatin. Compared with untreated mice, carboplatin-treated mice had a lower weight (23.3 ± 2.0 vs. 27.9 ± 2.9 g, p < 0.001), peritoneal carcinomatosis index (PCI, 11 ± 3 vs. 28 ± 6, p < 0.001), Ki67-positive staining surfaces (p < 0.001), vascular density (p < 0.001), mean blood flow velocity (mBFVel) in the SMA (7.0 ± 1.4 vs. 10.9 ± 1.8 cm/s, p < 0.001) and CT (8.0 ± 1.8 vs. 14.3 ± 4.6 cm/s, p < 0.001) and no ascites. Weight and mBFVel were similar in bevacizumab-treated and untreated mice. The mBFVels in the SMA and CT correlated with the PCI used as an estimation of the tumor burden, R = 0.70 (p < 0.0001) and R = 0.65 (p < 0.0001), respectively. Doppler US allows non-invasive assessment of the effects of anticancer therapy in ovarian peritoneal carcinomatosis-induced mice.

Chronic social defeat stress in female mice leads to sex-specific behavioral and neuroendocrine effects

Over the years, it has become increasingly clear that males and females respond differently towards environmental stressors, highlighting the importance of including both sexes when studying the effects of stress. This study aims to provide further insight into the detailed consequences of exposing female mice to 21 days of chronic social defeat stress (CSDS). We used a protocol that relies on the ability of odorants and pheromones in male urine to trigger male mouse aggressive behavior. Collected male C57Bl/6n urine was applied to female C57Bl/6n mice who were then attacked by a novel male CD1 mouse each day according to the CDSD protocol. Control females were pair-housed and handled daily. Physiological, neuroendocrine and behavioral changes were evaluated during the experiment. CSDS exposure resulted in number of physiological changes, such as body weight gain, enlarged adrenals and reduced thymus weight, exaggerated HPA-axis negative feedback and increased anxiety-like behavior. However, no generalized social avoidance behavior was observed. This study provides important insights in the physiological, neuroendocrine and behavioral responses of female mice to CSDS, which are partially dependent on estrous cycle stage. This protocol will allow direct comparison of male and female responses to CSDS and enable sex-specific study of mechanisms underlying individual stress resilience. Lay summary In this study we found that there are differences in the way that female and male mice respond towards chronic social stress conditions when it comes to behavior and hormonal changes.

Quercetin exerts antidepressant and cardioprotective effects in estrogen receptor α-deficient female mice via BDNF-AKT/ERK1/2 signaling

Brain-derived neurotrophic factor (BDNF) is the potential link between depression and cardiovascular disease and estrogen receptor α (ERα), an estrogen-mediated major regulator, plays an important role in protecting against depression and cardiovascular disease. However, the relationship between BDNF and ERα remains obscure. Herein, quercetin (QUE), a kind of plant flavonoids and existed in many vegetables and fruits, was found to simultaneously reverse ERα−/−-induced depression-like and cardiac dysfunction by reducing immobility time in the tail suspension test (TST) and forced swimming test (FST), and decreasing systolic blood pressure and activating the apoptosis-related proteins, BDNF, tropomyosin-related kinase B (TrkB), protein kinase B (AKT), and extracellular regulatory protein kinase (ERK1/2) in the hippocampal and cardiac tissues of female mice. These findings suggested that ERα might be involved in the regulation of BDNF activity, thereby regulating depression-like and cardiovascular responses in female mice, and QUE exerted significant antidepressant and cardioprotective effects, at least in part, through BDNF-TrkB-AKT/ERK1/2 to effectively inhibit ERα−/−-induced hippocampal and cardiac dysfunction.

Selective deletion of endothelial mineralocorticoid receptor protects from vascular dysfunction in sodium-restricted female mice

BackgroundRecent evidence by our laboratory demonstrates that women and female mice endogenously express higher endothelial mineralocorticoid receptor (ECMR) than males. Mounting clinical evidence also indicates that aldosterone production is higher in pathological conditions in females compared to males. However, the role for increased activation of ECMR by aldosterone in the absence of a comorbid condition is yet to be explored. The current study hypothesized that increased ECMR activation induced by elevated aldosterone production predisposes healthy female mice to endothelial dysfunction.MethodVascular reactivity was assessed in aortic rings from wild-type (WT) and ECMR KO (KO) mice fed either a normal salt (NSD, 0.4% NaCl) or sodium-restricted diet (SRD, 0.05% NaCl) for 28 days.ResultsSRD elevated plasma aldosterone levels as well as adrenal CYP11B2 and angiotensin II type 1 receptor (AT1R) expressions in female, but not male, WT mice. In baseline conditions (NSD), endothelial function, assessed by vascular relaxation to acetylcholine, was higher while vascular contractility to phenylephrine, serotonin, and KCl lower in female than male WT mice. SRD impaired endothelial function and increased vascular contractility in female, but not male, WT mice effectively ablating the baseline sex differences. NOS inhibition with LNAME ablated endothelial relaxation to a higher extent in male than female mice on NSD and ablated differences in acetylcholine relaxation responses between NSD- and SRD-fed females, indicating a role for NO in SRD-mediated endothelial function. In association, SRD significantly reduced vascular NOX4 expression in female, but not male, mice. Lastly, selective deletion of ECMR protected female mice from SRD-mediated endothelial dysfunction and increased vascular contractility.ConclusionCollectively, these data indicate that female mice develop aldosterone-induced endothelial dysfunction via endothelial MR-mediated reductions in NO bioavailability. In addition, these data support a role for ECMR to promote vascular contractility in female mice in response to sodium restriction.

Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.

Exposure to exertional heat stroke (EHS) has been linked to increased long-term decrements of health. Epigenetic reprogramming is involved in the response to heat acclimation; however, whether the long-term effects of EHS are mediated by epigenetic reprogramming is unknown. In female mice, we observed DNA methylation reprogramming in bone marrow-derived (BMD) monocytes as early as 4days of recovery from EHS and as late as 30days compared with sham exercise controls. Whole blood, collected after 30days of recovery from EHS, exhibited an immunosuppressive phenotype when challenged in vitro by lipopolysaccharide. After 30days of recovery from EHS, BMD monocytes exhibited an altered in vitro heat shock response. The location of differentially methylated CpGs are predictive of both the immunosuppressive phenotype and altered heat shock responses. Exposure to exertional heat stroke (EHS) has been linked to increased susceptibility to a second heat stroke, infection and cardiovascular disease. Whether these clinical outcomes are mediated by an epigenetic memory is unknown. Using a preclinical mouse model of EHS, we investigated whether EHS exposure produces a lasting epigenetic memory in monocytes and whether there are phenotypic alterations that may be consistent with these epigenetic changes. Female mice underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. Results were compared with matched exercise controls at 22.5°C. Monocytes were isolated from bone marrow after 4 or 30days of recovery to extract DNA and analyse methylation. Broad-ranging alterations to the DNA methylome were observed at both time points. At 30days, very specific alterations were observed to the promoter regions of genes involved with immune responsiveness. To test whether these changes might be related to phenotype, whole blood at 30days was challenged with lipopolysaccharide (LPS) to measure cytokine secretion; monocytes were also challenged with heat shock to quantify mRNA expression. Whole blood collected from EHS mice showed markedly attenuated inflammatory responses to LPS challenge. Furthermore, monocyte mRNA from EHS mice showed significantly altered responses to heat shock challenge. These results demonstrate that EHS leads to a unique DNA methylation pattern in monocytes and altered immune and heat shock responsiveness after 30days. These data support the hypothesis that EHS exposure can induce long-term physiological changes that may be linked to altered epigenetic profiles.

The Impact of Sex on Changes in Plasma Corticosterone and Cotinine Levels Induced by Nicotine in C57BL/6J Mice.

We assessed if there were any sex-related differences in the ability of nicotine to increase plasma corticosterone secretion after single or repeated nicotine administration. For single-dose studies, male and female mice were habituated to the test room for 1 h and injected with saline or nicotine (0.25 or 1 mg/kg, subcutaneously (s.c.)). In repeated-dosing studies, mice were injected with saline or nicotine (1 mg/kg, s.c.) once daily for six days, and, on day 7, received nicotine (1 mg/kg, s.c.). Mice were then euthanized 15 min later, and trunk blood was collected for the measurement of corticosterone, nicotine, and cotinine. Our results showed that saline or nicotine each significantly increased plasma corticosterone levels in both males and females, with a greater response in female mice. Plasma corticosterone levels were increased in male but not female mice after being treated repeatedly compared to single nicotine administration. The level of cotinine, a biomarker of nicotine use, was significantly higher in female than in male mice. Taken together, these novel findings suggest that female mice respond to nicotine and the stress of handling more than male mice and provide for the first-time quantitative data on male–female differences in nicotine-induced elevations of corticosterone and cotinine plasma levels.

Exacerbated obesogenic response in female mice exposed to early life stress is linked to fat depot-specific upregulation of leptin protein expression.

Early life stress (ELS) is an independent risk factor for increased BMI and cardiometabolic disease risk later in life. We have previously shown that a mouse model of ELS, maternal separation and early weaning (MSEW), exacerbates high-fat diet (HF)-induced obesity only in adult female mice. Therefore, the aim of this study was to investigate 1) whether the short- and long-term effects of HF on leptin expression are influenced by MSEW in a sex-specific manner and 2) the potential epigenetic mechanisms underlying the MSEW-induced changes in leptin expression. After 1 wk of HF, both MSEW male and female mice displayed increased fat mass compared with controls (P < 0.05). However, only MSEW female mice showed elevated leptin mRNA expression in gonadal white adipose tissue (gWAT; P < 0.05). After 12 wk of HF, fat mass remained increased only in female mice (P < 0.05). Moreover, plasma leptin and both leptin mRNA and protein expression in gWAT were augmented in MSEW female mice compered to controls (P < 0.05), but not in MSEW male mice. This association was not present in subcutaneous WAT. Furthermore, among 16 CpG sites in the leptin promoter, we identified three hypomethylated sites in tissue from HF-fed MSEW female mice compared with controls (3, 15, and 16, P < 0.05). These hypomethylated sites showed greater binding of key adipogenic factors such as PPARγ (P < 0.05). Taken together, our study reveals that MSEW superimposed to HF increases leptin protein expression in a sex- and fat depot-specific fashion. Our data suggest that the mechanism by which MSEW increases leptin expression could be epigenetic.

Distinct Alteration of Gene Expression Programs in the Small Intestine of Male and Female Mice in Response to Ablation of Intestinal Fabp Genes.

Fatty acid-binding proteins (Fabps) make up a family of widely distributed cytoplasmic lipid-binding proteins. The small intestine contains three predominant Fabp species, Fabp1, Fabp2, and Fabp6. Our previous studies showed that Fabp2 and Fabp6 gene-disrupted mice exhibited sexually dimorphic phenotypes. In this study, we carried out a systematic comparative analysis of the small intestinal transcriptomes of 10 week-old wild-type (WT) and Fabp gene-disrupted male and female mice. We found that the small intestinal transcriptome of male and female mice showed key differences in the gene expression profiles that affect major biological processes. The deletion of specific Fabp genes induced unique and sex-specific changes in the gene expression program, although some differentially expressed genes in certain genotypes were common to both sexes. Functional annotation and interaction network analyses revealed that the number and type of affected pathways, as well as the sets of interacting nodes in each of the Fabp genotypes, are partitioned by sex. To our knowledge, this is the first time that sex differences were identified and categorized at the transcriptome level in mice lacking different intestinal Fabps. The distinctive transcriptome profiles of WT male and female small intestine may predetermine the nature of transcriptional reprogramming that manifests as sexually dimorphic responses to the ablation of intestinal Fabp genes.

Life-long Dietary Pesticide Cocktail Induces Astrogliosis Along with Behavioral Adaptations and Activates p450 Metabolic Pathways

Exposure to environmental contaminants is a public health concern. However, pre-clinical studies that examine the impact of pesticides at low-dose and the long-term consequences are uncommon. Here, C57BL6/j male and female mice were daily fed from weaning and up to 12 months, corresponding to early-childhood into middle-age in humans, using chow pellets containing a cocktail of pesticides at tolerable daily intake levels. We found that 12 months of dietary exposure to pesticides was associated with a moderate perenchymal or perivascular astrogliosis in specific hippocampal sub-regions. The expression of platelet-derived growth factor receptor beta was modified at the perivascular level. Examination of Iba1+ microglial cells did not reveal sizeable changes. Concomitantly to astrogliosis, spontaneous spatial memory and sociability were modified in males at 12 months of dietary exposure to pesticides. Telemetry electrocorticograhic explorations ruled out the presence of epileptiform activity or theta-gamma wave modifications in these conditions. Long-term pesticides impacted the periphery where the hepatic P450 metabolic cytochromes Cyp4a14 and Cyp4a10 were significantly upregulated in male and female mice during the 12 months of exposure. The expression of β-oxidation genes, such as Acox1, Cpt1a and Eci, was also significantly increased in male and female mice in response to pesticides. Collectively, our results indicate that a life-long exposure to a pesticide cocktail elicits sex-dependent, spatio-temporally restricted brain modifications and significant activation of P450 pathways in the periphery. These brain-peripheral adjustments are discussed as time or age-dependent vulnerability elements.