Di-(2-ethylhexyl) phthalate (DEHP) is one of the most extensively used phthalate and poses a public health concern. Perinatal exposure to DEHP has been shown to cause neurodevelopmental abnormalities and neurobehavioral disorders in offspring. However, the precise molecular mechanism has not yet been fully elucidated. In this study, pregnant C57BL/6 mice were exposed to DEHP from gestation to weaning. By RNA sequencing and animal experiments, ferroptosis has been identified as the key pathologic process contributing to DEHP-induced hippocampal injury in adult male offspring. In vitro results also showed that Ferrostatin-1 (Fer-1) effectively ameliorated Mono-(2-ethylhexyl) phthalate (MEHP) -induced cell survival via the inhibiting ferroptosis in HT22 cells. Consistently, we found that the expression of ACSL4 and TFR was significantly up-regulated in offspring hippocampi and MEHP-exposed HT22 neurons. However, silencing ACSL4 or knockdown TFR relieved MEHP-induced generation of lipid ROS and cellular iron accumulation, thereby blocking ferroptosis. Mechanistically, ACSL4/TFR-mediated ferroptosis seemed to be a Yes-associated protein (YAP) dependent via TEA domain transcription factor 4 in HT22 neurons. Importantly, treatment with Fer-1, rosiglitazone, and Deferoxamine effectively rescued DEHP-evoked cognitive decline in adult male offspring. Our findings certified that gestational and lactational exposure to DEHP provoked ACSL4/TFR-mediated hippocampal neuronal ferroptosis via YAP activation.

Lateral ankle sprains (LAS) are associated with corticospinal pathway deficits. Existing evidence is primarily based on cross-sectional investigations and noncausal speculations. This study aims to determine whether maladaptive corticospinal pathway alterations occur pre- and postligament transection in LAS mouse models. Additionally, this study explores whether the alterations are more pronounced in adolescent mice than adults.

Twenty-four 8-week-old adolescent and twenty-four 24-week-old adult mice were randomly assigned to lateral ankle ligament transection or sham surgery. Diffusion-weighted imaging of the corticospinal pathway was performed presurgery and 8 weeks postsurgery. Fractional anisotropy (FA) values, reflecting fiber integrity within the corticospinal subregions of the medulla, pons, midbrain, and cerebrum, were extracted.

Overall, 41 mice completed repeated image acquisition. Before surgery, no significant group effects on FA within the four corticospinal subregions were detected in either adolescent or adult mice. Two months after surgery, the adolescent cohort displayed a significant reduction in FA in the medulla subregion following ankle ligament transection (β-baseline-adjusted ​= ​-0.083, 95% CI , -0.145 to -0.021, p-corrected ​= ​0.048). Conversely, no significant effects of ankle ligament transection on corticospinal FA were observed in the adult cohort.

The maladaptive alterations in the corticospinal tract could be observed in the adolescent LAS mouse model, characterized by reduced fiber integrity in the medulla subregion. While these results are derived from an animal model, they provide a foundation for future investigations into the mechanisms underlying neurological deficits following musculoskeletal injuries.

Anxiety and depression are two major contributors to global disease burden. Amongst various causal factors, exposure to even low doses of environmental heavy metals, like arsenic, can induce anxiety and depression-like behaviour in mammals. Ingestion of arsenic, primarily through contaminated drinking water, severely disrupts the gut microbes, thereby inducing structural and functional abnormalities in the brain. Fecal microbiota transplantation (FMT) from arsenic-exposed mice to recipient healthy mice (As-FMT) enriched LPS-secreting Gram-negative bacteria and upregulated the expression of TLR4 in intestinal epithelial cells. Consequently, inflammation, oxidative stress and compromised barrier integrity in the gut facilitated LPS translocation into the bloodstream and promoted systemic inflammation. The secretomes eventually affected the brain by activating microglia, altering neurotransmitter levels and reducing the glucocorticoid receptor (GR) expression, contributing to appearance of pyknotic nuclei in dentate gyrus of hippocampus and emergence of anxiety- and depression-like behaviour. Luteolin, a flavonoid, devoid of any apparent side-effects, yet known for its anti-inflammatory and antioxidant properties, showed potential in alleviating the gut-brain toxic effects. However, its limited solubility and bioavailability pose challenges for its effectiveness, for which chitosan-conjugated luteolin gold nanoparticles (CH-LuAuNPs) were synthesized. Interestingly, where FMT from arsenic-treated mice to healthy mice showed deleterious effects in the transplanted mice, FMT from arsenic-treated mice co-administered with CH-LuAuNP attenuated As-FMT-mediated disruption of the gut-brain axis. This study highlighted the critical contribution of healthy gut microbiota in preserving neurobehavioural physiology, as well as underscored the potential therapeutic benefits of luteolin nanoparticles in ameliorating arsenic-induced gut dysbiosis and consequent mental disorders.

The uterus is a hollow, fibromuscular organ involved in physiological processes such as menstruation and pregnancy. The content and organization of extracellular matrix constituents such as fibrillar collagen dictate passive (non-contractile) biomechanical tissue function; however, how extracellular matrix composition and biomechanical function change with age in the uterus remains unknown. This study utilizes Raman spectroscopy coupled with biaxial inflation testing to investigate changes in the murine uterus with age (2-3 months, 4-6 months, 10-12 months, and 20-24 months). Linear and toe moduli significantly decreased with reproductive aging (2 to 12 months); however, both moduli increased in the oldest age group (20-24 months). The optical concentration of the combined elastin and collagen spectrum was significantly higher in the oldest group (20-24 month), while the glycogen contribution was the highest in the 2-3 month murine uterus. The presented workflow couples biaxial inflation testing and Raman spectroscopy, representing a critical first step to correlating biomechanics and optical signatures in the aging uterus with the potential for clinical translation. Further, this study may provide critical compositional and structure-function information regarding age-related uterine disorders.

Gli1 has been identified as a marker of osteogenic progenitors in the condylar subchondral bone. The Wnt/β-catenin signaling pathway is known to regulate stem cell proliferation and differentiation in bone. Whether Wnt/β-catenin signaling pathway could influence Gli1 + osteogenic progenitors remains unclear. Here, we aimed to investigate the role and related mechanisms of Wnt/β-catenin signaling in the regulation of Gli1 + osteogenic progenitors in condylar development and temporomandibular joint osteoarthritis (TMJOA).

We generated Gli1-CreERT2;tdTomato mice to perform lineage tracing; We generated Gli1-CreERT2; β-cateninfl/fl mice, in which β-catenin was lost in the Gli1 + lineage to examine the role of Wnt/β-catenin signaling pathway in regulating the proliferation and differentiation of Gli1 + cells. The β-catenin CKO mice and their wild-type (WT) littermates were induced at 3 days and were euthanized 1, 2 or 4 weeks after induction; We induced a TMJOA model through a unilateral partial discectomy (UPD) of the temporomandibular joint disc in 6-week-old tamoxifen-treated Gli1-CreERT2;β-cateninfl/fl;tdTomato mice and control group (Gli1-CreERT2;tdTomato mice). We harvested the mandibles at 4 weeks post-surgery.

Conditional knockout of β-catenin inhibited the osteogenic activity of Gli1 + progenitor cells during condylar subchondral bone development. In discectomy-induced TMJOA, the overactivation of Gli1 in subchondral bone drove pathological osteogenesis and aberrant subchondral bone remodeling. Deletion of β-catenin in Gli1 + cells mitigated excessive Gli1 + cells activation and ectopic mineralization.

Our findings establish Wnt/β-catenin signaling as a key regulator of Gli1 + progenitor cell fate determination in both bone development and TMJOA pathogenesis.

Preeclampsia is a severe pregnancy complication associated with substantial injury to systemic vasculature, major organs, and the feto-placental unit, with an approximate mortality rate of 76,000 pregnant women and 500,000 babies each year. Preeclampsia results in up to five-fold increased risk of cardiovascular disease. There is currently no cure and limited treatment options for preeclampsia and its long-term effects. In this study, we modelled preeclampsia in the mouse via nitric oxide blockade and examined the effect of therapeutic intervention during pregnancy (esomeprazole) and postpartum (eplerenone) on indices of cardiovascular health. Pregnant CBA x C57BL/6 mice received 50 mg/kg/day N(ω)-nitro-L-arginine methyl ester to induce a preeclampsia-like phenotype. Mice were treated with either 12.5 mg/kg/day esomeprazole during pregnancy, 55.5 mg/kg/day eplerenone during the postpartum period, or both esomeprazole and eplerenone in sequence. Mice were hypertensive during pregnancy, fetal growth was restricted by 10%, and maternal vasoactivity was impaired at 5-weeks postpartum. Eplerenone treatment (± esomeprazole) reduced vasoconstriction at 5-weeks postpartum and enhanced vasorelaxation at 5- and 10-weeks postpartum, supporting improved cardiovascular indices in the medium to long term postpartum period. Postpartum eplerenone treatment may be beneficial in mitigating consequent cardiovascular disease risk following a pregnancy complicated by preeclampsia.

Single-cell proteomics is a pivotal technology for studying cellular phenotypes, offering unparalleled insights into cellular heterogeneity and dynamic functions. Technical improvement in mass spectrometry instruments and sample preparation has made proteomics profiling of single mouse oocytes or early embryos feasible in recent years. Yet, developing a simple and robust sample preparation method to enable deep proteomics profiling of single germline cells remains a significant challenge. Herein, we developed a simple one-step vial-based pretreatment (SOViP) for deep label-free single-cell proteomics of germline cells. SOViP integrates all sample preparation procedures into a single step in autosampler vials, yet it is highly efficient and high-throughput in comparison to reported multistep methods. SOViP can be finished within ∼2 h with hands-on time limited to merely a few minutes. On average, over 6500 protein groups can be quantified from a single mouse oocyte using SOViP. In total, 6983 protein groups were identified from single mouse oocytes across an entire reproductive lifespan, offering a valuable proteomics resource for oocyte aging. Unique molecular characteristics of oocytes at different ages were revealed, and a classifier consisting of ten proteins demonstrated accurate age-group classification and fertility-level prediction. Although demonstrated using mouse oocytes in this study, SOViP is adaptable to rare cell samples and other large cells including follicles and preimplantation embryo cells, among others.

Age-related osteoporosis poses a significant challenge in musculoskeletal health; a condition characterized by reduced bone density and increased fracture susceptibility in older individuals necessitates a better understanding of underlying molecular and cellular mechanisms. Emerging evidence suggests that osteocytes are the pivotal orchestrators of bone remodeling and represent novel therapeutic targets for age-related bone loss. Our study uses the prematurely aged PolgD257A/D257A (PolgA) mouse model to scrutinize age- and sex-related alterations in musculoskeletal health parameters (frailty, grip strength, gait data), bone and particularly the osteocyte lacuno-canalicular network (LCN). Moreover, a new quantitative in silico image analysis pipeline is used to evaluate the alterations in the osteocyte network with aging. Our findings underscore the pronounced degenerative changes in the musculoskeletal health parameters, bone, and osteocyte LCN in PolgA mice as early as 40 weeks, with more prominent alterations evident in aged males. Our findings suggest that the PolgA mouse model serves as a valuable model for studying the cellular mechanisms underlying age-related bone loss, given the comparable aging signs and age-related degeneration of the bone and the osteocyte network observed in naturally aging mice and elderly humans.

Epidemiological studies have demonstrated associations between heat waves, ozone (O3) pollution, and cardiovascular morbidity and mortality. High temperature (HT) and higher levels of O3 usually co-exist in the atmosphere. However, few studies have investigated the adverse effects of HT and O3 co-exposure on cardiovascular system. Therefore, this study aimed to examine the effects of HT and O3 co-exposure on biomarkers of cardiovascular damage and potential mechanisms. Sixty-four healthy SPF male C57BL/6N mice, aged 8 weeks, were randomly allocated into four groups: control, O3, HT, and co-exposure (HT+O3). Mice inhaled filtered air or 1 ppm O3 at 24 °C or 36 °C, respectively, 4 h/day, for 5 consecutive days. Following the exposure, the biological samples of mice were collected for examination of biomarkers of cardiovascular disorders. Exposure to HT+O3 exacerbated cardiovascular pathological damage induced by HT or O3 alone. Compared to the control, the co-exposure group caused significant alterations of cardiovascular biomarkers. Moreover, co-exposure enhanced reduction of Lactobacillus and Ruminococcus and increases in Prevotella and Alistipe abundances induced by either HT or O3. Moreover, co-exposure also promoted O3-induced plasma metabolic disorder and these metabolites were enriched in multiple metabolic pathways typified by steroid hormone biosynthesis, biosynthesis of unsaturated fatty acids, and phenylalanine metabolism, among others. Spearman correlation analysis indicated that alterations of gut microbiota were significantly correlated with biomarkers of cardiovascular damage as well as plasma metabolic disorder. Exposure to HT and O3 leads to cardiovascular damage, which possibly implicates gut microbial dysbiosis and plasma metabolic disorder.

X-ALD is a white matter (WM) disease caused by mutations in the ABCD1 gene encoding the transporter of very-long-chain fatty acids (VLCFAs) into peroxisomes. Strikingly, the same ABCD1 mutation causes either devastating brain inflammatory demyelination during childhood or, more often, progressive spinal cord axonopathy starting in middle-aged adults. The accumulation of undegraded VLCFA in glial cell membranes and myelin has long been thought to be the central mechanism of X-ALD.

This review discusses studies in mouse and drosophila models that have modified our views of X-ALD pathogenesis.

In the Abcd1 knockout (KO) mouse that mimics the spinal cord disease, the late manifestations of axonopathy are rapidly reversed by ABCD1 gene transfer into spinal cord oligodendrocytes (OLs). In a peroxin-5 KO mouse model, the selective impairment of peroxisomal biogenesis in OLs achieves an almost perfect phenocopy of cerebral ALD. A drosophila knockout model revealed that VLCFA accumulation in glial myelinating cells causes the production of a toxic lipid able to poison axons and activate inflammatory cells. Other mouse models showed the critical role of OLs in providing energy substrates to axons. In addition, studies on microglial changing substates have improved our understanding of neuroinflammation.

Animal models supporting a primary role of OLs and axonal pathology and a secondary role of microglia allow us to revisit of X-ALD mechanisms. Beyond ABCD1 mutations, pathogenesis depends on unidentified contributors, such as genetic background, cell-specific epigenomics, potential environmental triggers, and stochasticity of crosstalk between multiple cell types among billions of glial cells and neurons.

To investigate how Porphyromonas gingivalis induces endothelial dysfunction, focusing on the regulatory role of Sirtuin 3 (Sirt3) in mitochondrial function.

Differentially expressed Sirtuin family genes in P. gingivalis-infected human aortic endothelial cells (HAECs) were identified through RNA sequencing and validated by quantitative real-time PCR and Western blot. Mitochondrial and endothelial functions were assessed in P. gingivalis-infected HAECs with or without Sirt3-specific agonist Honokiol. Cyclophilin D (CypD) K167 point mutation plasmids were constructed, and Co-immunoprecipitation was performed to investigate the Sirt3-CypD interaction. The vasorelaxation of aortas from mice orally administrated with P. gingivalis was also evaluated.

Porphyromonas gingivalis infection in HAECs resulted in mitochondrial and endothelial dysfunction. Mechanistic studies revealed that Sirt3-mediated deacetylation of CypD at K167 was pivotal in alleviating P. gingivalis-induced mitochondrial and endothelial dysfunction. Oral inoculation of P. gingivalis in mice significantly impaired endothelial-dependent vasodilation, disrupted aortic endothelial integrity, increased endothelial cell apoptosis, and elevated mitochondrial reactive oxygen species production. Notably, Sirt3 activation reversed mitochondrial and endothelial dysfunction induced by P. gingivalis both in vivo and in vitro.

The present study demonstrated that P. gingivalis induced mitochondrial and endothelial dysfunction, which was mediated through Sirt3-dependent CypD deacetylation.

Background/Objectives: The loss of retinal ganglion cells (RGCs) is a hallmark of glaucoma, a major cause of blindness. Glial cell activation due to increased intraocular pressure (IOP) significantly contributes to RGC death. Therefore, substances with anti-inflammatory properties could help prevent that process. This study investigated whether combining Citicoline and Coenzyme Q10 (CoQ10) can reduce glial activation in the retina and the rest of the visual pathway, potentially preventing neurodegeneration in a mouse model of unilateral laser-induced ocular hypertension (OHT). Methods: Four groups of mice were used: vehicle (n = 12), CitiQ10 (n = 12), OHT-vehicle (n = 18), and OHT-CitiQ10 (n = 18). The administration of Citicoline and CoQ10 was performed orally once a day, initiated 15 days prior to the laser treatment and maintained post-treatment until sacrifice (3 days for retina or 7 days for the rest of the visual pathway). The retina, dorsolateral geniculate nucleus, superior colliculus, and visual cortex (V1) were immunohistochemically stained and analyzed. Results: In the laser-CitiQ10 group, the Citicoline + CoQ10 compound revealed (1) an IOP decrease at 24 h and 3 days post-laser; and (2) reduced signs of macroglial (decreased GFAP area) and microglial (soma size, arbor area, microglia number, P2RY12 expression) activation in the retina and in the rest of the visual pathway (reduced activated microglial phenotypes and lower GFAP expression). Conclusions: This study shows that oral administration of Citicoline and CoQ10 can reduce glial activation caused by increased IOP in retina and visual pathway in a mouse model of OHT, potentially protecting RGCs from OHT-induced inflammation.

Cognitive impairment associated with natural aging significantly reduces the healthy lifespan of elderly adults. Barley is rich in polysaccharides, particularly starch and dietary fibers such as β-glucan and xylan. As the predominant components of barley water extracts, these polysaccharides, especially dietary fibers, exhibit substantial potential in promoting gut and brain health. In this study, we established a natural aging model by exposing mice to a high-fat diet and chronic stress for 220 consecutive days. Our findings revealed that barley polysaccharides ameliorated cognitive deficits, particularly long-term memory, by modulating neurotransmitter levels and reducing corticosterone. Barley polysaccharides also alleviated lipid metabolism disorders, reduced liver lesions, and decreased body weight as well as the percentage of visceral fat in mice by regulating bile acid and l-lysine metabolism. Additionally, barley polysaccharides enhanced intestinal barrier integrity and reshaped the gut microbiota. They significantly increased the abundance of norank_f_Muribaculaceae and unclassified f_Lachnospiraceae, leading to elevated short-chain fatty acid levels, especially butyric acid, which contributed to improved cognitive function. These findings suggest that barley polysaccharides could serve as a promising dietary intervention to mitigate cognitive decline associated with natural aging through the liver-gut-brain axis.

Mouse models are widely used to establish new therapy concepts for acute lung injury, but the transfer of therapeutic approaches into the intensive care unit often failed. To establish a mouse intensive care unit to adequately reflect the patient's situation and to investigate sex- and age-related differences in response to lipopolysaccharide.

For the establishment of a mouse intensive care unit, young (2-3 months) and old (15-18 months) mice of both sexes received continuous respiratory and cardiovascular monitoring for 6 h. Mimicking an acute lung injury by intratracheal lipopolysaccharide stimulation for 6 or 24 h, the impact of sex and age on survival and physiological parameters was evaluated.

The establishment revealed sex- and age-related differences in physiological responses during mechanical ventilation, with old males requiring more noradrenaline to maintain stable hemodynamics. While young mice, irrespective of sex, developed acute lung injury 24 h after lipopolysaccharide administration, old mice exhibited a rapid systemic response, showing signs of lactic acidosis and endotoxemia. Among these, old females had the highest mortality risk, whereas in old males, mechanical ventilation provided effective support, contributing to improved survival outcomes.

We successfully established a mouse intensive care unit that integrated all critical aspects of a human intensive care unit simultaneously. By highlighting sex- and age-related differences following lipopolysaccharide stimulation and mechanical ventilation, our study underscored the need for diversity in preclinical models to improve translation of findings on critical illnesses like acute lung injury into clinical settings.

Frailty syndrome, a condition marked by increased vulnerability due to age-related physiological decline, exerts a profound impact on skeletal muscle structure and function. Despite its widespread prevalence, the underlying mechanisms contributing to frailty-associated muscle deterioration remain poorly elucidated. This study utilized histological and biochemical analyses in a murine model to investigate the effects of frailty syndrome on skeletal muscle. Mice were classified based on age and condition, including a subset subjected to an exercise intervention. Parameters evaluated included body weight, lean mass ratio, myofiber size and number, extracellular matrix (ECM) content, and myosin heavy chain isoform expression. Frailty syndrome led to increased body weight and ECM content, coupled with reductions in myofiber size and number, reflecting substantial structural and functional impairments in skeletal muscle. Exercise interventions effectively countered these deleterious changes, preserving myofiber morphology and reducing ECM expansion, thereby demonstrating the protective role of exercise in mitigating frailty-induced muscle deterioration. The study highlights the severe impact of frailty syndrome on skeletal muscle structure and integrity. Importantly, it underscores the potential of regular exercise as an effective therapeutic approach to prevent or reverse muscle deterioration associated with frailty, offering critical insights into managing age-related muscular degeneration.

Aging is a major risk factor for cardiovascular disease, with hypertension being the most common outcome. Hypertension often stems from resistance arteries endothelial dysfunction. Recent research highlights the pivotal role of perivascular adipose tissue (PVAT) in regulating endothelial function. We hypothesized that PVAT senescence contributes to vascular dysfunction and hypertension during aging. We showed that naturally aged mice developed hypertension and elevated pro-inflammatory cytokines levels. Moreover, resistance mesenteric arteries showed impaired vascular relaxation that was normalized by apocynin, an antioxidant. The vascular dysfunction was endothelium- and PVAT-dependent, and marked by: decreased nitric oxide- and cyclooxygenase-dependent vascular relaxation, decreased expression of endothelial nitric oxide synthase, and increased cyclooxygenase 2 and NADPH oxidase subunits p22phox and gp91phox expressions in the endothelium and PVAT. Additionally, we observed that PVAT shows greater signs of senescence, particularly with higher p16 expression, indicating that PVAT is more prone to age-related cellular aging. Our findings suggest that in resistance mesenteric arteries PVAT-derived factors are crucial for triggering and amplifying vascular dysfunction in aging, leading to hypertension. The underlying mechanisms involve downregulation of endothelial nitric oxide synthase-derived nitric oxide, NADPH oxidase-dependent oxidative stress, and cyclooxygenase 2-derived vascular contractile factors. This research improves our understanding of the mechanisms behind age-related vascular dysfunction and associated hypertension and opens perspectives for targeted therapeutic strategies.

Primary sarcopenia, an age-related syndrome, is a serious threat to the health and longevity of the elderly. Our prior studies indicated that thyroid hormone (TH) activity within muscle tissue undergoes significant age-associated alterations, mainly evidenced by a reduction in thyroid hormone receptor α (TRα) expression over time. TRα regulates the transcription of downstream target genes to exert its biological effects. Although TH is essential for skeletal muscle growth and development, the specific regulatory mechanism and broader role of TH binding its receptors in skeletal muscle aging remain unclear. We used ChIP-seq and RNA-seq to explore the aging changes of TRα target genes in gastrocnemius muscle of natural aging mouse model. ChIP-seq analysis revealed that TRα target genes are involved in nutrient synthesis, energy production, hormone secretion, and ECM-related pathways, suggesting a potential role of TRα in muscle growth, metabolism and component regulation. Further integration of RNA-seq showed that a greater number of down-regulated TRα target genes are associated with skeletal muscle aging. Through GSEA analysis and RT-qPCR screening, Col6a1 was identified as a key target gene. Col6a1 encodes collagen VI which is an important component of the ECM, ECM disorders and abnormal expression of Col6a1 can affect cell proliferation and differentiation. We confirmed that knockdown of Col6a1 inhibited the proliferation and differentiation of C2C12 cells. ChIP-qPCR and TRα silencing in C2C12 cells showed that TRα positively regulates Col6a1 transcription, and TRα deficiency inhibits the proliferation and differentiation of myoblasts, which is probably associated with Col6a1. These findings provide new insights into the molecular mechanisms underlying skeletal muscle aging and the regulatory roles of TH-TRα interactions.

Preterm infants born in the saccular stage of lung development are at risk for developing bronchopulmonary dysplasia (BPD). Oxygen toxicity and volutrauma are identified as major contributors of BPD. Despite mitigation of these risks preterm infants continue to be affected by chronic lung disease. Heparin is commonly administered to preterm infants and is known to interfere with angiogenesis, a critical element of lung development. We previously demonstrated, in a murine model, that compensatory lung growth after left pneumonectomy is inhibited by heparin administration. Based on these results, we hypothesized that heparin would interfere with lung development in neonatal mice, which are born during the saccular phase of lung development. Newborn C57BL/6J mice received either therapeutic unfractionated heparin (UFH), low molecular weight heparin (LMWH) or normal saline (control) for the first week of life. At one month, both UFH and LMWH produced an emphysematous lung phenotype. Late administration of heparin, after the saccular phase did not impact lung function or growth. This data establishes the negative effects of UFH and LMWH during the critical period of postnatal lung development. Based on this work, clinical studies on the impact of heparin on lung development of newborn and preterm infants are warranted.

Maternal inflammation during pregnancy increases the offspring's risk of developing autism, ADHD, schizophrenia, and depression. Epidemiologic studies have demonstrated that maternal infections stimulate the production of interleukin-6 (IL-6), which can cross the placenta and fetal blood-brain barrier to alter brain development with functional and behavioral consequences. To model the effects of increased IL-6 between weeks 24-30 of human gestation, we injected male and female mice with 75 ng IL-6 twice daily, from P3 to P6. Our published studies have shown that this increases circulating IL-6 two-fold, alters post-pubescent ultrasonic vocalization patterns, reduces sociability, and increases self-grooming. However, most neurodevelopmental disorders in humans manifest in children as young as 2 years of age. Hence, a critical unexplored question is whether behavioral changes in immune activation models can be detected in pre-pubescent mice. Therefore, we evaluated early communication, sociability, and repetitive behaviors in pre-pubescent mice following the IL-6 treatment. A second open question is whether the cellular and behavioral changes are secondary to systemic or neuroinflammation. To address this question, we profiled 18 cytokines and chemokines in the circulation and CNS and evaluated eight immune cell types in P7 male and female brains following systemic IL-6 administration. We found an increase in ultrasonic vocalizations with simpler morphologies produced by the IL-6-injected male pups and a decrease in frequency in the female vocalizations upon removal from the nest at P7. The IL-6-treated male pups also socially interacted less when introduced to a novel mouse vs. controls as juveniles and spent almost twice as much time grooming themselves, a phenotype not present in the females. Tactile sensitivity was also increased, but only in the IL-6-treated female mice. The IL-6-treated mice had increased circulating IL-6 and IL-7 and reduced IL-13 at P7 that were no longer elevated at P14. There were no changes in brain levels of IL-6, IL-10, IL-13 or IL-17A mRNAs at P7. Altogether, these studies show that changes in the three core behavioral domains associated with several psychiatric disorders can be detected early in pre-pubescent mice following a transient developmental increase in IL-6. Yet, these behavioral alterations do not require neuroinflammation.

Alzheimer's disease (AD), a neurodegenerative disorder with progressive cognitive decline, remains clinically challenging with limited understanding of etiology and interventions. Clinical studies have reported vascular defects prior to other pathological manifestations of AD, leading to the "Vascular Hypothesis" for the disorder. However, in vivo assessments of cerebral vasculature in AD rodent models have been constrained by limited spatiotemporal resolution or field of view of conventional imaging. We herein employed two in vivo imaging technologies, Dual-Wavelength Imaging and Optical Coherence Doppler Tomography, to evaluate cerebrovascular reactivity (CVR) to vasoconstrictive cocaine and vasodilatory hypercapnia challenges and to detect resting 3D cerebral blood flow (CBF) in living transgenic AD mice at capillary resolution. Results showed that CVR to cocaine and hypercapnia was significantly attenuated in 7-10 months old AD mice vs controls, indicating reduced vascular flexibility and reactivity. Additionally, in the AD mice, arterial CBF velocities were slower and the microvascular density in cortex was decreased compared to controls. These results reveal significant vascular impairments including reduced CVR and resting CBF in early-staged AD mice. Hence, this cutting-edge in vivo optical imaging offers an innovative venue for detecting early neurovascular dysfunction in AD brain with translational potential.

Decades of research into the function of the medial temporal lobe has driven curiosity around clinical outcomes associated with hippocampal dysfunction, including psychosis. Post-mortem analyses of brain tissue from human schizophrenia brain show decreased expression of the NMDAR subunit GluN1 confined to the dentate gyrus with evidence of downstream hippocampal hyperactivity in CA3 and CA1. Little is known about the mechanisms of the emergence of hippocampal hyperactivity as a putative psychosis biomarker. We have developed a reverse-translation mouse to study critical neural features. We had previously studied a dentate gyrus (DG)-specific GluN1 KO, which displays hippocampal hyperactivity and a psychosis-relevant behavioral phenotype. Here, we expressed an inhibitory DREADD (pAAV-CaMKIIa-hM4D(Gi)-mCherry) in granule cells of the mouse dentate gyrus, and continuously inhibited the region for 21 days in adolescent (6 weeks) and adult (10 weeks) C57BL/6 J mice with DREADD agonist Compound 21 (C21). Following this period, we quantified activity in the hippocampal subfields by assessing cFos expression, hippocampally mediated behaviors, and hippocampal local field potential with an intracerebral probe with continual monitoring over time. DG inhibition during adolescence generates an increase in hippocampal activity in CA3 and CA1, impairs social cognition and spatial working memory, as well as shows evidence of increased activity in local field potentials as spontaneous synchronous bursts of activity, which we term hyper-synchronous events (HSEs) in hippocampus. The same DG inhibition delivered during adulthood in the mouse lacks these outcomes. These results suggest a sensitive period in development in which the hippocampus is susceptible to DG inhibition resulting in hippocampal hyperactivity and psychosis-like behavioral outcomes.

For a potential resource to improve healthspan, polysaccharides present unique advantages in terms of side effects and long-term use owing to their low cytotoxicity. In this study, we demonstrate that a glucomannogalactan (PGP) derived from Pleurotus geesteranus extends the healthspan of both naturally senescent and therapy-induced senescence (TIS) mice. Daily treatment of naturally senescent mice with PGP resulted in a reduced accumulation of senescent cells and alleviation of senescence-related parameters, including metabolic dysfunction, underlying lesions in multiple organs, and oxidative damage. PGP treatment also attenuated senescence in TIS mice. Furthermore, in an in vitro model of oxidative stress-induced senescence using a human cell line, we discovered that PGP alleviated senescence by promoting the nuclear translocation of NRF2. This study suggests that PGP may extend the healthspan of senescent mice by facilitating the nuclear translocation of NRF2.

The beneficial effects of dietary fiber for colon health may be due to short chain fatty acids (SCFAs), such as butyrate, produced by colonic bacterial fermentation. In contrast, obesogenic diet induced obesity is linked to increased colon cancer incidence. We hypothesize that increasing fiber intake promotes healthy microbiome and reduces bacterial dysbiosis and oncogenic signaling in the colon of mice fed an obesogenic diet. About 5-week-old male C57BL/6 mice were assigned to 5 dietary groups (n=22/group) for 24 weeks:(1) AIN93G as a control diet (AIN); (2) a high fat diet (HFD, 45% energy fat); (3) HFD+5% resistant starch enriched dietary fiber (RSF) from maize; (4) HFD+10%RSF; or (5) HFD+20%RSF. Compared to the AIN group, mice receiving the HFD exhibited more than 15% increase in body mass and body fat composition irrespective of RSF dosage. However, the HFD+RSF groups exhibited an increase (>300%) of fecal butyrate but a decrease (>45%) of secondary bile acids in a RSF dose-dependent manner over the HFD group. Similarly, there were concomitant decreases (>25%) in pro-inflammatory plasma cytokines (TNFα, IL-6 and MCP-1), β-catenin and Ki67 protein staining in the colon of the HFD+20%RSF group relative to the HFD group. Furthermore, the abundance of colonic Proteobacteria, signatures of dysbiosis, was decreased (>63%) in a RSF dose-dependent manner compared to the HFD. Collectively, these data indicate that RSF not only increases butyrate but also reduces secondary bile acids, bacterial dysbiosis and β-catenin in the colon of mice fed a HFD.

Advanced paternal age constitutes a noteworthy risk factor for neurodevelopmental disorders in progeny, encompassing conditions such as schizophrenia, autism, and atypical social behavior. Nonetheless, the precise underlying mechanisms remain inadequately comprehended. In this study, we delved into the alterations of DNA methylation induced by aging in sperm and pre-implantation embryos of male mice. A total of 3909 Differentially Methylated Regions (DMRs) were identified in sperm from aged male mice compared to young group. In addition, the overall DNA methylation in androgenetic 2-cell embryos exhibited a pronounced increase in aged group, leading to altered expression of genes related to neuropsychiatric disorders. Interestingly, a total of 242 shared DMRs were identified through the overlap of the sperm DMR sets and the 2-cell embryo DMR sets. This finding suggests the plausible involvement of these shared DMRs in the intergenerational transmission of epigenetic traits. Furthermore, F1 male mice from aged group exhibited a marked decrease in sociability and displayed DNA methylation alterations associated with nerve signal transduction components in their brain tissues. Intriguingly, inhibition of DNA methyltransferases (DNMT3A/3B) by siRNA in pre-implantation embryos improved abnormal social behaviors in F1 males from aged fathers, with concomitant changes in the expression of genes related to nerve development detected in 4-cell embryos. Our study indicates that male aging exert an influence not only on DNA methylation modification in sperm and pre-implantation embryo, but also on neurobehavioral abnormalities of their offspring. Repairing DNA methylation in pre-implantation embryos may offer a promising avenue for ameliorating abnormal social behavior in offspring.

Amyloid accumulation in Alzheimer's disease (AD) is associated with synaptic damage and altered connectivity in brain networks. While measures of amyloid accumulation and biochemical changes in mouse models have utility for translational studies of certain therapeutics, preclinical analysis of altered brain connectivity using clinically relevant fMRI measures has not been well developed for agents intended to improve neural networks. Here, we conduct a longitudinal study in a double knock-in mouse model for AD (AppNL-G-F/hMapt), monitoring brain connectivity by means of resting-state fMRI. While the 4-month-old AD mice are indistinguishable from wild-type controls (WT), decreased connectivity in the default-mode network is significant for the AD mice relative to WT mice by 6 months of age and is pronounced by 9 months of age. In a second cohort of 20-month-old mice with persistent functional connectivity deficits for AD relative to WT, we assess the impact of two-months of oral treatment with a silent allosteric modulator of mGluR5 (BMS-984923/ALX001) known to rescue synaptic density. Functional connectivity deficits in the aged AD mice are reversed by the mGluR5-directed treatment. The longitudinal application of fMRI has enabled us to define the preclinical time trajectory of AD-related changes in functional connectivity, and to demonstrate a translatable metric for monitoring disease emergence, progression, and response to synapse-rescuing treatment.

The onset of multiple sclerosis (MS) in older individuals correlates with a higher risk of developing primary progressive MS, faster progression to secondary progressive MS, and increased disability accumulation. This phenomenon can be related to age-related changes in the immune system: with age, the immune system undergoes a process called immunosenescence, characterized by a decline in the function of both the innate and adaptive immune responses. This decline can lead to a decreased ability to control inflammation and repair damaged tissue. Additionally, older individuals often experience a shift toward a more pro-inflammatory state, known as inflammaging, which can exacerbate the progression of neurodegenerative diseases like MS. Therefore, age-related alterations in the immune system could be responsible for the difference in the phenotype of MS observed in older and younger patients. In this study, we investigated the effects of age on the immunopathogenesis of experimental autoimmune encephalomyelitis (EAE). Our findings indicate that EAE is more severe in aged mice due to a more inflammatory and neurodegenerative environment in the central nervous system. Age-related changes predominantly affect adaptive immunity, characterized by altered T cell ratios, a pro-inflammatory Th1 response, increased regulatory T cells, exhaustion of T cells, altered B cell antigen presentation, and reduced NK cell maturation and cytotoxicity. Transcriptomic analysis reveals that fewer pathways and transcription factors are activated with age in EAE. These findings allow us to identify potential therapeutic targets specific to elderly MS patients and work on their development in the future.

The present study aimed to assess whether Time-Restricted Feeding (TRF) modulates inflammation and hepatic Notch1 signalling in C57BL/6J-aged mice.

Adult mice submitted to the ad libitum diet, aged (24 months-old) submitted to the ad libitum diet and, aged-TRF (24 months-old) subjected to the TRF (12 hours fed in the active cycle and 12 hours fasting in the light cycle) for 8 weeks. We investigated metabolic parameters, liver histology, metabolic-dysfunction-associated fatty liver disease activity score, collagen fiber, hepatic mitochondrial respiration, and publicly available liver Rna-seq datasets from human livers in diverse clinical conditions to clarify Notch1 involvement in liver health.

Our results demonstrated that aged mice (24 months old) showed increases in body weight, liver mass, Notch1 intracellular domain (NICD), and inflammatory markers (NFκB and TLR4 protein levels) in the liver when compared to adult animals. On the other hand, aged mice submitted to a TRF protocol showed reductions in inflammation and collagen fibers, which was accompanied by lower protein content of JAGGED1 and NICD in the liver. Furthermore, aged-TRF mice demonstrated increased liver mitochondrial respiration coupled with ATP production compared to the aged groups. Publicly available liver RNA-seq datasets in humans support our findings, indicating the upregulation of NOTCH1 in fibrosis and inflammation development.

TRF can reduce inflammatory markers and protein content of JAGGED1 and NICD in the liver of aged mice, which can contribute to tissue health and cellular longevity.

During aging, protein nutrition has a bidirectional role in regulating healthy lifespan by modulating body metabolism and neurological function. However, the current "low-high" hypothesis on the dynamics of protein requirements is mainly based on male animal models, and its applicability to female physiology (e.g., estrogen fluctuations) is unclear. The present study aims to fill the gap in the study of protein demand dynamics in female naturally aging mice and to investigate the effects of different protein levels on the health status of female C57BL/6J mice at different stages of aging.

In this study, four dietary interventions (high protein, HP; low protein, LP; model test, MT; and control, C) were evaluated by constructing a C57BL/6J female mouse model at three ages, 9 M (9 months), 16 M (16 months), and 20 M (20 months), which are approximately equivalent to 34, 65, and 78 years of age in humans, respectively, to determine the effects on naturally aging mice. The effects of the interventions were quantitatively described by behavioral, neuropathological, oxidative, and inflammatory indices and NMR metabolomics using Principal Component Analysis to construct a comprehensive quantitative scoring method.

The comprehensive quantitative scores Fsum was highest in the HP group, lowest in the LP group, and in between in the MT group. The HP intervention showed the most significant improvement in the aged group (20 M) mice, with a 35.2% reduction in avoidance latency (p < 0.01) and a 32.9% increase in pyramidal cell density in the hippocampal CA1 region (p < 0.05), while the LP intervention led to a cognitive decline in the mice, with an avoidance latency that was prolonged by 15.2% (p < 0.05). Metabolomics analysis revealed that mouse samples of all ages showed age-dependent metabolic re-adaptation: the 9 M group may reflect gut microbial metabolism rather than direct host TCA cycle activity, suggesting an indirect association with energy metabolism; an enhanced degradation of branched-chain amino acids (BCAAs) was seen in the middle-aged group (16 M); and amino acid biosynthesis was predominant in the old group (20 M).

Female mice have sustained neuromotor benefits to high-protein diets at different aging stages, and the dynamics of their protein requirements differ significantly from those of males. The study reveals the critical role of gender factors in protein nutritional strategies and provides an experimental basis for precise protein supplementation in older women.

There is an escalating need to comprehend the long-term impacts of nuclear radiation exposure since the permeation of ionizing radiation has been frequent in our current societal framework. A system evaluation of the microbes that reside inside a host's colon could meet this knowledge gap since the microbes play major roles in a host's response to stress. Indeed, our past study suggested that these microbes might break their symbiotic association with moribund hosts to form a pro-survival condition exclusive to themselves. In this study, we undertook metagenomics and metabolomics assays regarding the descending colon content (DCC) of adult mice. DCCs were collected 1 month and 6 months after 7 Gy or 7.5 Gy total body irradiation (TBI). The assessment of the metagenomic diversity profile in DCC found a significant sex bias caused by TBI. Six months after 7.5 Gy TBI, decreased Bacteroidetes were replaced by increased Firmicutes in males, and these alterations were reflected in the functional analysis. For instance, a larger number of networks linked to small chain fatty acid (SCFA) synthesis and metabolism were inhibited in males than in females. Additionally, bioenergy networks showed regression dynamics in females at 6 months post-TBI. Increased accumulation of glucose and pyruvate, which are typical precursors of beneficial SCFAs coupled with the activated networks linked to the production of reactive oxygen species, suggest a cross-sex energy-deprived state. Overall, there was a major chronic adverse implication in male mice that supported the previous literature in suggesting females are more radioresistant than males. The sex-biased chronic effects of TBI should be taken into consideration in designing the pertinent therapeutics.

Background/Objectives: Prostate cancer (PC) is among the most common malignancy in men. Several newly diagnosed patients have a locally advanced disease and distant metastasis at the initial diagnosis time. Castration-resistant PC (CRPC) patients have 100% recurrence incidence despite completing a therapeutic regimen, leading to high mortality. Androgen deprivation therapy and androgen inhibitors are initially effective, but resistance is inevitably developed. Epidemiological studies indicated that the Mediterranean diet, with high olive phenolic contents, is associated with a lower incidence of certain malignancies. This study aims at exploring the mCRPC progression and recurrence-suppressive and molecular effects of the major olive leaf phenolic glucoside (-)-oleuropein (OLE). Results: OLE downregulated the levels of proprotein convertase subtlisin/klexin type 9 (PCSK9) and normalized the low-density lipoprotein receptor (LDLR) in PC cells in vitro. Thus, a PCSK9-LDLR protein-protein interaction (PPI) in silico model was generated and used to assess OLE and its aglycone (OA) ability to bind at PCSK9 and thereby interfere with PCSK9-LDLR PPI. OLE perfectly filled the PCSK9 interface versus OA. Both OLE and OA showed virtual potential to interfere with PCSK9-LDLR PPI. OLE showed modest in vitro viability, migration, and clonogenicity suppressive effects on diverse human PC cell lines. OLE effectively suppressed mCRPC progression and recurrence in a nude mouse xenograft model. RNA-sequencing results proved the PCSK1, PCSK2, and PCSK9 downregulation in OLE-treated recurrent tumors versus vehicle control. Conclusions: Oleuropein is a novel lead useful for the control of mCRPC progression and the prevention of its recurrence via targeting PCSK9 expression and PPI with LDLR.

Leishmaniasis is a complex disease caused by protozoan parasites of the genus Leishmania, which are transmitted by phlebotomine sand flies. The clinical manifestations of leishmaniasis are diverse, ranging from self-healing cutaneous lesions to fatal systemic disease. Mouse models are instrumental in advancing our understanding of the immune system against infections, yet their limitations in translating findings to humans are increasingly highlighted. The success rate of translating data from mice to humans remains low, largely due to the complexity of diseases and the numerous factors that influence the disease outcomes. Therefore, for the effective translation of data from murine models of leishmaniasis, it is essential to align experimental conditions with those relevant to human infection. Factors such as parasite characteristics, vector-derived components, host status, and environmental conditions must be carefully considered and adapted to enhance the translational relevance of mouse data. These parameters are potentially modifiable and should be carefully integrated into the design and interpretation of experimental procedures in Leishmania studies. In the current paper, we review the challenges and perspective of using mouse as a model for leishmaniasis. We have particularly emphasized the non-genetic factors that influence experiments and focused on strategies to improve translational value of studies on leishmaniasis using mouse models.

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) is an X-linked rare neurodevelopmental disorder associated with severe sleep disturbances. However, little is known about the mechanisms underlying sleep disturbances in CDD patients. Here, we employed the electroencephalogram (EEG) recording to characterize sleep-wake behaviors and EEG activity in male CDKL5-deficient mice. We found that young adult and middle-aged Cdkl5 knockout (KO) mice recapitulated sleep phenotypes in patients with CDD, including difficulties in initiating and maintaining sleep, reduction in total sleep time, and frequent night awakenings. Cdkl5 KO mice exhibited pre-sleep arousal, but normal circadian rhythm and homeostatic sleep response. Conditional knockout (cKO) of Cdkl5 in glutamatergic neurons resulted in reduced sleep time and difficulty in sleep maintenance. Further, the rate of age-associated decline in sleep and EEG activity in Cdkl5 KO mice was comparable to that of wild-type littermates. Together, these results confirm a causative role for CDKL5 deficiency in sleep disturbances observed in CDD patients and establish an animal model for translational research of sleep treatment in CDD. Moreover, our results provide valuable information for developing therapeutic strategies and identifying sleep and EEG parameters as potential biomarkers for facilitating preclinical and clinical trials in CDD.

Statins are one of the top prescribed medications and are used for preventing or treating cardiovascular diseases. Myalgia, muscle fatigue, weakness, and inflammation are the most common side effects of these drugs collectively named statin-associated muscle symptoms (SAMS). The mechanisms underlying SAMS remain unclear. Given that statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of mevalonate pathway, responsible for synthesis of cholesterol and other vital molecules, SAMS may be mediated by multiple reasons. Herein, using unbiased whole transcriptome sequencing, we identified statin-affected processes and then assessed them using fluorescent, biochemical, and histological approaches in the mouse diaphragm, the main respiratory muscle. Mice were orally treated for 1 month with atorvastatin, the most prescribed statin, at clinically relevant dose. We found that atorvastatin caused downregulation of genes encoding proteins required for oxidative phosphorylation and anabolic processes, whereas genes of proteins engaged inflammation and muscle atrophy were mainly up-regulated. Furthermore, alterations in gene expression pattern suggest oxidative stress and abnormal lipid accumulation. This transcriptome signature correlated to a decrease in mitochondrial polarization and protein synthesis capacity, as well as an increase in lipid peroxidation and reactive oxygen species production. In addition, atorvastatin treatment caused lipid raft disruption, phospholipidosis, myelin de-compactization, and appearance of greater heterogeneity of muscle fiber cross-section diameter. Thus, atorvastatin treatment can negatively affect diaphragm muscle via oxidative stress accompanied by decrease in mitochondrial activity, protein synthesis, and stability of plasma membrane. As a part of compensatory response can serve enhanced activity of superoxide dismutase and cholesterol uptake capacity.