3-month Mice Research Articles

Exercise evaluation with metabolic and ventilatory responses and blood lactate concentration in mice

This study aimed to clarify the differential exercise capacity between 2-month-old and 10-month-old mice using an incremental running test. Metabolic and ventilatory responses and blood lactate concentration were measured to evaluate exercise capacity. We examined whether incremental running test results reflected metabolic and ventilatory responses and blood lactate concentration observed during the steady-state running test. Metabolic response significantly declined with age, whereas ventilatory response was similar between the groups. A low-intensity/moderate exercise load of 10/min in an incremental running test was performed on both mice for 30 min. They showed a characteristic pattern in ventilatory response in 10-month mice. The results of incremental running tests didn’t necessarily reflect the steady-state metabolic and ventilatory responses because some parameters showed an approximation and others did not in incremental and steady-state tests, which changed with age. Our study suggests metabolic and ventilatory responses depending on age and provides basic knowledge regarding the objective and quantitative assessment of treadmill running in an animal model.

Electroacupuncture ameliorates postoperative cognitive dysfunction and associated neuroinflammation via NLRP3 signal inhibition in aged mice.

BackgroundPostoperative cognitive dysfunction (POCD) is associated with worsened prognosis especially in aged population. Clinical and animal studies suggested that electroacupuncture (EA) could improve POCD. However, the underlying mechanisms especially EA’s regulatory role of inflammasomes remain unclear.MethodsThe model of POCD was established by partial hepatectomy surgery in 18‐month mice with or without postoperative EA treatment to the Baihui acupoint (GV20) for 7 days. Cognitive functions were assessed by Morris water maze test, and proinflammatory cytokines IL‐1β and IL‐6 and microglia activity were assayed by qPCR, ELISA, or immunohistochemistry. Tight junction proteins, NLRP3 inflammasome and downstream proteins, and NF‐κB pathway proteins were evaluated by western blotting.ResultsEA markedly preserved cognitive dysfunctions in POCD mice, associated with the inhibition of neuroinflammation as evidenced by reduced microglial activation and decreased IL‐1β and IL‐6 levels in brain tissue. EA also preserved hippocampal neurons and tight junction proteins ZO‐1 and claudin 5. Mechanistically, the activation of NLRP3 inflammasome and NF‐κB was inhibited by EA, while NLRP3 activation abolished EA’s treatment effects on cognitive function.ConclusionEA alleviates POCD‐mediated cognitive dysfunction associated with ameliorated neuroinflammation. Mechanistically, EA’s treatment effects are dependent on NLRP3 inhibition.

LOAD2: A late‐onset Alzheimer’s disease mouse model expressing APOEε4 , Trem2*R47H , and humanized amyloid‐beta

Alzheimer's disease (AD) is the most common form of dementia without an approved therapy. Current animal models do not fully recapitulate late-onset AD (LOAD), thus are not ideal for therapy development. The Model Organism Development and Evaluation for Late-onset AD (MODEL-AD) Center is developing and distributing novel mouse models carrying human-relevant risk factors. Characterization of aging mice that better phenocopy human disease will reveal more appropriate disease pathways useful in the treatment of LOAD.Humanized amyloid-beta (Aβ) in addition to APOEε4 and Trem2*R47H, two risk factors of LOAD, were incorporated into C57BL/6J mice to produce the triple homozygous LOAD2 model. Cohorts of mice were aged on multiple sites to 4-, 12-, 18-, and 24-month timepoints. In some mice, high-fat diet replaced normal mouse chow. A phenotyping pipeline designed to qualify disease states was implemented to measure behavior, cognition, and wellness. In vivo imaging of brain perfusion and metabolism, is also included, as is post-mortem analyses of blood chemistry, neuropathology, transcriptomics, metabolomics, proteomics, spatial profiling, and electrophysiology.Expression of nonmutated Aβ did not yield in 18-month mice. By 12 months, LOAD2 mice did not display penetrant behavioral phenotypes. High-fat diet increased frailty scores in all cohorts irrespective of genotype. LOAD2 mice show increased plasma cytokines and neuroinflammation in 12-month animals without overt hippocampal pathology. Long-term potentiation is preserved in LOAD2 animals until 12-months. Correlation of transcriptional profiling with human AMP-AD modules determined individual and synergistic effects between genetic and environmental risk factors. Neuropathology and in vivo imaging analysis of brain tissue is in progress, as are cytokine panels in brain homogenates and plasma.The MODEL-AD consortium has established the LOAD2 mouse model to study the effects of genetic and environmental risk factors of LOAD. This strain serves as a platform for the incorporation of additional AD risk factors (both genetic and environmental to more closely align phenotypes in the mouse to outcomes observed in the clinic. Ultimately, strains carrying combinations of AD risk factors that more closely align with human disease will be incorporated into the pre-clinical testing core of MODEL-AD to assess the potential of prioritized therapeutic compounds.

SIRT3 retards intervertebral disc degeneration by anti-oxidative stress by activating the SIRT3/FOXO3/SOD2 signaling pathway.

The objective of this paper is to determine whether SIRT3 could retard intervertebral disc degeneration and study the mechanism. We chose the 3-month mice to establish intervertebral disc degeneration model and study the effect of SIRT3 on the intervertebral disc by Western blotting, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), immunohistochemistry. Mouse nucleus pulposus cells were cultured to study the exact mechanism. The expression of SIRT3 was decreased in degenerated human nucleus pulposus. Intervertebral discs of mice treated with theacrine expressed more collagen II and less collagen X. In addition, nucleus pulposus cells stimulated with interleukin-1β (IL-1β) expressed less SIRT3 than that in the control group and nucleus pulposus cells with SIRT3 overexpress vectors expressed more collagen II FOXO3a and superoxide dismutase 2 (SOD2), indicating that SIRT3 could improve the intervertebral disc degeneration by anti-oxidative stress. SIRT3 is a protective factor for intervertebral discs and can reduce oxidative stress in the intervertebral disc.

The aging effects on phenylephrine-induced relaxation of bladder in mice.

Objective:We have demonstrated that phenylephrine (PE) activates the capsaicin-sensitive nerves, and then activates capsaicin-sensitive nerves to release an unknown substance that facilitates the release of norepinephrine (NE) from adrenergic nerves. Subsequently, NE stimulates β-ARs in the detrusor muscle in mice, leading to neurogenic relaxation of the urinary bladder (UB).Materials and Methods:We examined if there existed sensory-motor dysfunction in UB of aging mice. To investigate the change of PE-induced detrusor relaxation in aging male-C57BL/6 mice (12- vs. 24-month-old mice), UB strips from mice were isolated, cut into strips, and mounted in the organ bath.Results:The UB strip contractility responding to various agents was estimated using tissue bath wire myography. Acetylcholine (ACh) and KCl-induced UB strips contraction was not significantly different between 24- and 12-month mice. NE-induced UB strips relaxation was significantly lower in 24-month than 12-month mice. Denuded bladder strips showed similar decreased relaxation response to NE. This NE-induced relaxation was inhibited by silodosin and lidocaine. PE did not induce contraction in UB strips of aging mice. In contrast, PE-induced relaxation was weaker in 24-month than 12-month mice.Conclusion:Our results suggested that the PE-induced relaxation was age related. Aging seemed to lead the sensory-motor dysfunction. More animal and human studies are required to prove this concept and its clinical usefulness in the future.

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.

Effects of prodynorphin deletion on striatal dopamine in mice during normal aging and in response to MPTP

Dynorphins, endogenous neuropeptides found in striatonigral neurons, have been observed to exhibit dopamine-inhibitory actions and under some circumstances possess intrinsic neurotoxic activity. To test the hypothesis that dynorphin suppression mitigates effects of aging on the striatal dopaminergic system, HPLC quantitation of dopamine and related amines was performed on striatal homogenates of wild-type (WT) mice and mice lacking the prodynorphin ( Pdyn) gene at varying ages. Pdyn knockout (KO) mice at 10 and 20 months show significant elevations in striatal dopamine compared to 3-month mice. Differences in tyrosine hydroxylase (TH) immunoreactivity could not account for these findings, but phosphorylation of TH at Ser40, but not Ser31, was enhanced in aged Pdyn KO mice. Systemic administration of MPTP produced significant dopamine depletion in an age-dependent manner, but Pdyn deletion conferred no protection against MPTP-induced dopamine loss, arguing against a mechanism by which Pdyn deletion enhances dopaminergic neuron survival. The above findings demonstrate an age-dependent inhibitory effect of dynorphins on striatal dopamine synthesis via modulation of TH activity.

Development of Mice Biomagnetic Measurement System Using dc-SQUID Magnetometer

A biomagnetic measurement system on mice using a dc superconducting quantum interference device magnetometer has been developed. We are going to use it for comparative magnetoencephalogram (MEG) and magnetocardiogram (MCG) studies of transgenic mice to clarify the functional influence of gene modification in a living body. The system has a low-T/sub c/ direct coupled dc SQUID. A focuser type pickup coil whose outside/inside diameter is 1 mm/100 /spl mu/m was adopted for detailed functional mapping of the brain or the heart of mice. The minimum lift-off distance was reduced to 700 /spl mu/m to achieve the sufficient spatial resolution and magnetic field sensitivity. The field sensitivity of this system is 1.3 pT/Hz/sup 1/2/ in the white-noise region (10 Hz-10 kHz). The MCG of a wild mouse at 9-months of age was measured and compared with the data of 3-month old mice. The spatial and time changes of MCG signals were similar in both ages of mice, while the amplitude of MCG signals of 9-month mice was less than 40% of that of 3-month mice. This decrease in MCG amplitude may be due to aging. The result suggested the capability of our system in evaluating the mice MCG signals.

Prodynorphin knockout mice demonstrate diminished age-associated impairment in spatial water maze performance

Dynorphins, endogenous κ-opioid agonists widely expressed in the central nervous system, have been reported to increase following diverse pathophysiological processes, including excitotoxicity, chronic inflammation, and traumatic injury. These peptides have been implicated in cognitive impairment, especially that associated with aging. To determine whether absence of dynorphin confers any beneficial effect on spatial learning and memory, knockout mice lacking the coding exons of the gene encoding its precursor prodynorphin ( Pdyn) were tested in a water maze task. Learning and memory assessment using a 3-day water maze protocol demonstrated that aged Pdyn knockout mice (13–17 months) perform comparatively better than similarly aged wild-type (WT) mice, based on acquisition and retention probe trial indices. There was no genotype effect on performance in the cued version of the swim task nor on average swim speed, suggesting the observed genotype effects are likely attributable to differences in cognitive rather than motor function. Young (3–6 months) mice performed significantly better than aged mice, but in young mice, no genotype difference was observed. To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus and frontal cortex of WT 129SvEv mice. Quantitative radioimmunoassay demonstrated that dynorphin A levels in frontal cortex, but not hippocampus, of 12- and 24-month mice were significantly elevated compared to 3-month mice. Although the underlying mechanisms have yet to be elucidated, the results suggest that chronic increases in endogenous dynorphin expression with age, especially in frontal cortex, may adversely affect learning and memory.

Acute radiolabeling of retinoids in eye tissues of normal and rpe65-deficient mice.

Mice with a targeted disruption of the gene encoding RPE65, a protein ordinarily highly expressed in the retinal pigment epithelium (RPE), accumulate abnormally high levels of all-trans retinyl ester in the RPE and exhibit very little 11-cis retinal in the retina. The present study was undertaken to determine whether the Rpe65-deficient mouse exhibits an abnormal flux of retinoid between the systemic circulation and the eye tissues. Dark-adapted Rpe65-deficient mice (Rpe65(-/-)) and wild-type control mice (Rpe65(+/+)) of approximate ages 1 and 3 months received an intraperitoneal injection of all-trans ((3)H)retinol. The mice were maintained in darkness for a defined period ( approximately 1.5, 4.5, 24, or 48 hours) and then anesthetized, exsanguinated, and killed. Retinoids contained in the retina, RPE, serum, and liver were extracted and analyzed for ((3)H) radioactivity and molar level. The specific activity (SA, in counts per minute per nanomole) of serum all-trans ((3)H)retinol in all mice exhibited a peak at postinjection times of 1.5 or 4.5 hours, and by 48 hours declined to approximately 7% or less of the peak. In Rpe65(+/+) mice, the average SA of RPE ((3)H)retinyl ester similarly exhibited an early peak (4.5 hours) and by 48 hours declined to approximately 6% to 10% of the peak. By contrast, the average SA of RPE ((3)H)retinyl ester in Rpe65(-/-) mice exhibited a peak at 24 or 48 hours. Radioactivity and molar data for serum all-trans retinol and RPE retinyl ester obtained at 4.5 hours were analyzed to infer the molar influx of all-trans retinol from the circulation into the RPE. Levels of all-trans retinol influx derived from this analysis (mean +/- SD: 0.014 +/- 0.004 nmol in 1-month Rpe65(+/+) mice; 0.021 +/- 0.009 nmol in 1-month Rpe65(-/-) mice; 0.016 +/- 0.013 nmol in 3-month Rpe65(+/+) mice; 0.026 +/- 0.018 nmol in 3-month Rpe65(-/-) mice) did not differ significantly from one another (P > 0.169). However, the inferred fractional influx (molar amount of entering all-trans retinol divided by the molar amount of RPE retinyl ester) in Rpe65(+/+) animals (0.34 +/- 0.04 and 0.10 +/- 0.03, respectively, for 1- and 3-month mice) substantially exceeded that for Rpe65(-/-) animals (0.055 +/- 0.023 and 0.015 +/- 0.006, respectively, for 1- and 3-month mice). Significant levels of ((3)H)retinaldehydes were detected in the retinas of Rpe65(+/+) mice, but not in those of Rpe65(-/-) mice, after the longer postinjection periods. The results indicate preservation of a substantial inward flux of all-trans retinol from the circulation into the RPE of Rpe65(-/-) mice, despite the presence of abnormally high molar levels of RPE retinyl ester. They further imply the occurrence of a robust outward movement of all-trans retinol from the RPE into the circulation in Rpe65(+/+) mice, and substantial impairment of this efflux process in Rpe65(-/-) mice. These findings raise the hypothesis that in normal RPE, 11-cis retinal and/or 11-cis retinol stimulate the efflux of all-trans retinol at the RPE basolateral membrane. In 3-month Rpe65(+/+) mice, the observed relationship between the SAs of retinaldehydes in the retina and of RPE retinyl ester is consistent with a last-in/first-out processing of all-trans retinol to 11-cis retinal within normally functioning RPE.

Age-related changes in benzene disposition in male C57BL/6N mice described by a physiologically based pharmacokinetic model

A physiologically based pharmacokinetic (PBPK) model was developed to describe the disposition of benzene in 3- and 18-month C57BL/6N mice and to examine the relevant physiologic and/or biochemical parameters governing previously observed age-related changes in the disposition of benzene. The model developed was based on that of Medinsky et al. (Toxicol. Appl. Pharmacol. 99 (1989) 193–206), with the inclusion of an additional rate constant for urinary elimination of benzene metabolites. Experimentally determined tissue partition coefficients for benzene in 3- and 18-month mice, as well as actual body weights and fat compartment volumes, were included as part of the model. Model simulations were conducted for oral exposure of 3-month mice to 10 and 200 mg benzene/kg and for oral exposure