Search Results (631)

Cold exposure is an unavoidable stressor in cold regions, leading to growth retardation, oxidative damage, and endocrine disruption. This study investigated changes in blood parameters and antioxidant function in the brown adipose tissue (BAT) of mice exposed to cold. Sixteen naturally mated female mice (aged 70 days) were selected and divided into a control group (CON, n = 8, 25 ± 1 °C) and a cold exposure group (CE, n = 8, 4 ± 1 °C). Each pregnant female gave birth to approximately 12 pups, and the litter (dams and pups co-housed) served as the independent experimental unit, with both euthanized for sampling when the pups reached 20 days of age. Results showed that cold exposure increased ADFI and ADG but decreased the feed conversion rate (FCR) in lactating mice. It also decreased platelet count (PLT) and mean corpuscular hemoglobin concentration (MCHC), elevated lactate dehydrogenase (LDH) activity, and decreased TG and non-esterified fatty acid (NEFA) levels. Hormonal changes included increased adrenocorticotropic hormone (ACTH), apelin 12 (AP12), INS, NE, decreased cortisol (COR), LEP, and thyroid-stimulating hormone (TSH). In pups, cold exposure inhibited growth, reduced PLT, plateletcrit (PCT), red blood cells (RBC), and hemoglobin (HGB), altered lipid profiles, and induced hormonal shifts. Notably, cold exposure enhanced the BAT antioxidant capacity in pups, increasing the total antioxidant capacity (T-AOC) and antioxidant enzyme activities, as supported by gene expression. These findings suggest that, despite growth suppression, mice maintain homeostasis by modulating blood parameters and enhancing BAT antioxidant function to mitigate cold-induced damage. Full article

Background/Objectives: Neuritogenesis and synaptogenesis support learning and cognitive function, and hippocampal neurons play central roles in these processes. Cleistocalyx nervosum var. paniala (CNP), a Southeast Asian berry, has reported neuroprotective activities, but its direct effects on hippocampal neurons remain unclear. We investigated whether CNP extract modulates hippocampal neuronal transcriptomes, neuritogenesis, and synaptogenesis. Methods: Primary hippocampal neurons isolated from male and female Wistar rat pups were treated with CNP extract in vitro. Cytotoxicity was assessed to define non-cytotoxic concentrations. Transcriptomic responses were profiled by RNA sequencing and validated by RT-qPCR. Neuritogenesis was quantified by neurite morphology and Sholl analysis. Synaptogenesis was evaluated by synaptic immunocytochemistry. Molecular docking of cyanidin-3-glucoside (C3G) and resveratrol was used to generate mechanistic hypotheses. Results: At 0.1–10 µg/mL, CNP was non-cytotoxic, whereas a 100 µg/mL dose reduced viability; therefore, 10 µg/mL was used in subsequent experiments. Exploratory RNA-seq profiling identified thousands of differentially expressed genes enriched in synapse- and neurite-related pathways, including synaptogenesis signaling, axon guidance, and neuritogenesis. RT-qPCR showed upregulation of Igf1 in males and Glul in females, with sex-dependent modulation of Bdnf and Cask. CNP increased neurite length, branching, and Sholl complexity in both sexes, with a more pronounced effect in males. A male-biased effect was also observed in synapse-related marker colocalization, with increased Syn1–Psd95 colocalization detected in males. Docking suggested plausible interactions of C3G and resveratrol with regulators such as MYC, TP53, and CREB1. Conclusions: CNP extract alters transcriptional networks and enhances neurite outgrowth in primary hippocampal neurons in a sex-dependent manner, with male-biased effects on Syn1–Psd95 colocalization. These findings support further dose–response, mechanistic, and sex-stratified in vivo studies to evaluate its neurobiological potential. Full article

Unbiased stereology represents the most accurate approach for estimating the total number of neurons of specific brain regions; however, its reliability critically depends on the use of rigorously defined and anatomically appropriate sampling parameters. The brain nucleus Locus Coeruleus (LC) plays a key role in several brain functions. LC impairment has been associated with a range of disorders affecting individuals across the lifespan, from infancy to adulthood. In animal models of these conditions, precise estimation of LC neuronal number is essential. The LC analysis poses specific methodological challenges due to its small size, indistinct anatomical boundaries, and age-dependent changes in neuronal density. In this study, we present a detailed and reproducible stereological workflow for the quantification of LC neurons in the mouse brain across the lifespan. Using C57BL/6J mice at postnatal, adult, and aged stages, we optimized all key components of the Optical Fractionator method, LC neurons were identified by immunoperoxidase staining for tyrosine hydroxylase (TH) and quantified using systematic-random sampling implemented in Stereo Investigator^® software. We show that age-specific adjustment of stereological parameters is necessary to obtain reliable estimates, particularly at early postnatal stages characterized by high neuronal packing density. With the optimized protocols described here, TH+ LC neuron counts consistently met accepted precision criteria, as assessed by the Gundersen coefficient of error. Full article

Background/Objectives: The vibrotactile system, which is essential for guiding behavior in nocturnal rodents such as mice and rats, provides critical sensory input. To investigate the role of vibrotactile sensory inflow in neonatal locomotion, we used previsual rat pups that underwent bilateral vibrissectomy. Subsequently, their motor behavior was evaluated in an open field test. Methods: A total of 42 previsual pups from four litters were assigned to either bilateral vibrissectomy or sham surgery groups on postnatal days (PND) 9–12, with group allocation balanced across litters. Results: Open-field testing on PND 13 revealed that while vibrissectomy (VE) did not affect gross locomotor activity—such as distance traveled, speed, acceleration, or freezing episodes (all >0.05)—it significantly altered spatial behavior. To quantify spatial patterns of curvy tracks, we analyzed trajectorial compaction within the central zone, lacking the tactile guidance of the walls: trajectories were smoothed using virtual coatings scaled to the vibrissal length (16 mm). For each track, an individual linearized reference path was generated and subjected to identical smoothing. The compaction ratio—calculated as the coated area of the smoothed linearized reference divided by the coated area of the experimental track—was significantly greater in VE pups than in sham controls (p = 0.03). This effect was not attributable to differences in the path length traveled within the central zone. The increased compaction persisted when the smoothing scale was increased 2–3 fold (32–64 mm radii, approximating the pups’ mean body size), but not at smaller scales (2–4 mm). Conclusions: These results demonstrate that tactile input specifically modulates the spatial, rather than locomotor, components of nonvisual navigation. Consequently, the track compaction may serve as a sensitive marker for assessing vibrotactile function in developing laboratory rodents. Full article

Well-founded animal models of maternal separation show that early postnatal separation from the mother represents a massive stress factor for the offspring, with far-reaching negative effects on their physical and behavioural development. However, studies also show that the effects of maternal separation can be at least mitigated by appropriate physical attention. In this case report, we document the successful rehabilitation of a single orphaned male harbour seal pup by attempting to substitute natural maternal attention with intensive human attention. The pup underwent excellent physical development and displayed a close bond with its carers, characterised by intensive physical contact and suckling on a carer’s hand. However, during the final rehabilitation phase in a large outdoor enclosure, the pup increasingly reduced its affiliation to its carers. After its release, long-term observations over four years documented its hunting success, corresponding optimal physical development, and successful integration into existing wild seal groups. At the age of four, the now sub-adult seal displayed socio-sexual behaviour that is typical for this age and maintained a close bond with a wild female seal for months. The report shows that the rehabilitation of orphaned seals is possible by substituting maternal care with human care, resulting in neither a fixation on humans nor modifying species-specific behaviour. Thus, the pup’s chances of survival and potential reproduction in the wild were not jeopardised by this method of rehabilitation. Full article

The intestinal barrier undergoes profound changes with age, impacting local immunity and systemic health, yet the mechanisms coordinating immune and microbial dynamics across the lifespan remain incompletely understood. Toll-like receptor 4 (TLR4) serves as a key mediator of host–microbiota interactions. This study investigated age-related changes in barrier function and the role of TLR4 using C57BL/6J and TLR4 knockout (TLR4^−/−) mice across key developmental stages: pups (postnatal day 9), adults (2–4 months), middle-aged (7–9 months), and old (16–19 months). Through a multi-layered approach integrating histology, microbiome profiling, short-chain fatty acid (SCFA) analysis, cytokine quantification, ex vivo functional assays, and transcriptomics, we identified a multi-phase process of intestinal remodeling. Pup-P9 mice exhibited immature colonic structure, a simple microbiota dominated by Firmicutes and Proteobacteria, and undetectable acetic acid level. Adults reached peak diversity and SCFA concentrations, marked by a rise in Bacteroidota and the emergence of Akkermansia. In middle and old age, pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) increased, Bacteroidota declined while Firmicutes, Actinobacteria, and Turicibacter expanded, and aged colons showed blunted ex vivo responses to IL-1β. This age-associated functional decline phenotype was absent in TLR4^−/− mice, supporting the involvement of TLR4 signaling. Transcriptomics further revealed biphasic PI3K/Akt activation in both pups-P9 and old mice. Together, these findings suggest a systemic rewiring of host metabolic and immune signaling pathways in response to an aging microbiota, highlighting this dynamic, lifespan-wide microbiota–host signaling axis as a potential intervention target. Full article

Normal pregnancy is associated with uterine and vascular remodeling by matrix metalloproteinases (MMPs) to facilitate placental blood flow and uterine expansion for the growing fetus. Increases in MMP-2 and MMP-9 in response to estrogen and progesterone promote placentation, uteroplacental vascularization and fetal growth during healthy pregnancy, but are altered in preeclampsia (PE). PE is characterized by hypertension in pregnancy (HTN-Preg) and fetal growth restriction (FGR). Predisposing genetic, demographic and environmental factors alter uteroplacental MMPs, immune response and integrins leading to apoptosis of invasive trophoblasts, inadequate spiral arteries remodeling, and reduced uteroplacental perfusion pressure (RUPP). Ensuing placental ischemia causes imbalance between anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and pro-angiogenic placental growth factor (PlGF) and promotes the release of tumor necrosis factor-α (TNF-α), hypoxia-inducible factor, reactive oxygen species, and angiotensin AT₁ receptor agonistic autoantibodies. Systemically, these bioactive factors target vascular endothelial cells, smooth muscle cells, and extracellular matrix, causing endothelial dysfunction, vasoconstriction, inadequate vascular remodeling, and HTN-Preg, while locally they diminish uteroplacental remodeling and cause FGR. In support, animal models of HTN-Preg induced by RUPP or infusion of sFlt-1 or TNF-α show decreases in vascular MMP-2, MMP-9 and vasodilation, increases in MMP-1, MMP-7 and vasoconstriction, collagen accumulation, and arterial stiffness. Also, decreases in uterine MMP-2 and MMP-9 could impede uterine expansion and lead to preterm birth. Conversely, PlGF and TNF-α antagonist reversed MMPs imbalance and collagen accumulation, and improved vascular function, blood pressure, and pup weight in HTN-Preg models. Persistent postpartum changes in MMPs could affect maternal hemorrhage, future pregnancies, and HTN, and cause fetal programming of cardiovascular and metabolic diseases. Understanding the aberrant uteroplacental and vascular signaling and remodeling by MMPs could help design new biomarkers and remedies for PE. Targeting bioactive factors and rectifying MMP imbalance could improve vascular and uteroplacental remodeling, and manage HTN-Preg, FGR and PE. Full article

Genome stability is the cornerstone of cellular health, and imbalances can cause a number of outcomes, including aging, cancer, and other pathologies. DNA damage is a strong driver of both cellular senescence and mitochondrial dysfunction, two other key hallmarks of aging. Both nuclear and mitochondrial genome instability have been shown to drive aging in the hematopoietic system, which then propagates to non-lymphoid tissues, enhancing morbidity and mortality. The loss of TFAM, a key regulator of mitochondrial DNA replication and nucleoid stability, in T cells has been shown to cause mitochondrial dysfunction, leading to premature immune aging and eventual systemic aging. We sought to investigate whether the loss of TFAM in all immune cells would have a comparable or stronger effect on both the immune system and parenchyma. To address this, we attempted to generate Vav-iCre^+/−; Tfam^fl/fl mice, which are deficient in TFAM in all immune cells. However, this genotype was unrecoverable as no mutant pups were born, suggesting embryonic lethality. Conversely, we generated mice lacking SIRT6, a nuclear DNA repair enzyme that also regulates mitochondrial homeostasis, in all immune cells and found them to be viable and born at expected Mendelian frequencies. Our findings demonstrate the necessity of mitochondrial genome maintenance and homeostasis repair in immunity. Full article

Purpose: To investigate the mechanisms by which plasma extracellular vesicles (EVs) from preterm infants with bronchopulmonary dysplasia (BPD) elicit inflammation and abnormal angiogenesis in neonatal mouse retinas. Methods: EVs from the plasma of 7-day-old preterm infants, born between 23^0/7 and 29^6/7 weeks of gestation, with BPD or without BPD (nBPD) at 36 weeks postmenstrual ages, were adoptively transferred into postnatal day 3 (P3) mice via intravenous retro-orbital sinus injection. Inflammation and pathological neovascularization in neonatal mouse retinas were examined by immunohistochemistry of retinal flat mounts for Allograft Inflammatory Factor 1 (AIF1), CD206, or Glial Fibrillary Acidic Protein (GFAP) and isolectin-B4 (IB4) staining on P17. Retinal inflammation-related transcripts were assessed by qRT-PCR. Proteomic profiles of BPD and nBPD EVs were examined by Liquid Chromatograph Mass Spectrometer/Mass Spectrometer (LC-MS/MS) and Gene Set Enrichment Analysis (GSEA). Results: Adoptively transferred EVs from BPD and nBPD infants crossed the blood–retinal barrier (BRB) in recipient mouse pups. BPD-EVs increased retinal activated microglia, Müller cells, and twisted proliferative neovascularization compared to nBPD-EVs. BPD-EVs also elevated retinal transcripts regulating inflammation and angiogenesis, including NOD-, LRR- and pyrin domain-containing protein 3 (Nlrp3), Apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), Caspase 3 (Casp3), Caspase 8 (Casp8), Gasdermin D (Gsdmd), Il1β, Il6, Aif1, and Vascular endothelial growth factor (Vegf). Proteomics analysis revealed that BPD-EVs had significantly elevated levels of inflammation and angiogenesis-related proteins compared to nBPD-EVs. Conclusions: BPD-EVs promote inflammation and abnormal neovascularization by upregulating genes related to apoptosis and inflammation in neonatal mouse retinas. EV protein profiles suggest that elevated levels of proteins such as Defensin alpha 1B (DEFA1B), Insulin-like growth factor binding protein 2 (IGFBP2), CD5 antigen-like (CD5L), von Willebrand factor (vWF), and Tenascin C (TNC) in BPD-EVs may contribute to the observed inflammation and angiogenesis. Full article

Epidemiological data link a lack of breastfeeding with an increased risk of breast cancer. Breast tissue remodels after pregnancy through involution. Long-term breastfeeding results in gradual involution (GI), and a lack of breastfeeding leads to abrupt involution (AI). AI causes increased mammary gland estrogen signaling, causing adipocyte redifferentiation through neutrophil infiltration. Adipocyte differences and metabolic implications of involution have not been explored between AI and GI. As breast cancer is characterized as highly metabolic, we explored how adipocyte differences and metabolism during involution may support breast cancer risk. FVB/n was randomized to AI/GI and standardized to 6 pups on day 0/birth. AI mice had pups removed on day 7. GI mice had 3 pups removed on days 28 and 31. Mammary glands were harvested at 28, 56, and 120 days. A subset of AI mice were given tamoxifen for 21 days. Day 28 AI glands had upregulation of estrogen signaling, neutrophil degranulation, and glucose metabolism and downregulation of adipogenesis and glycolysis compared to Day 56 GI. Day 120 AI glands had downregulation of oxidative phosphorylation and upregulation of mitochondrial dysfunction similar to estrogen receptor-negative (ER−) pregnancy-associated breast cancer (PABC). AI with tamoxifen resulted in a similar metabolic phenotype to GI. Early metabolic phenotypes in AI and GI glands may be related to estrogen signaling. AI long-term transcriptional metabolic effects were similar to breast cancer. Full article

Background: Environmental agents can disrupt thyroid function at several levels, including the synthesis, action, and excretion of thyroid hormones, and an inadequate concentration of thyroid hormones affects almost all organs and systems. Objective: The present study aimed to evaluate the histology and presence of the cytokines TNFα, IL-6, and IL-10 in the thyroid gland by immunohistochemical labeling, as well as the body weight and craniocaudal length of pups of Wistar rats exposed to ambient air in the vicinity of the Capuava Petrochemical Complex (CPC), located in the Santo André and Mauá cities, at São Paulo State, Brazil. Methods: This study used Wistar rats between 14 and 16 weeks of age, distributed in couples, that were exposed to pollution from the CPC located in the regions of Santo André and Mauá, Sao Paulo State, Brazil. One couple was positioned 600 m (SS1), and another was 1000 m (SS2) from the CPC, while the control group was kept at the animal research facility of the Physiology Laboratory of the FMABC University Center, Santo André. After mating, the resulting offspring were monitored for four weeks, with their body weight and craniocaudal length measured weekly. Subsequently, the offspring’s thyroid glands were histologically analyzed using H&E staining and immunohistochemistry to detect the presence of inflammatory cytokines (TNF-α, IL-6, and IL-10). Results: In the SS1 group, the thyroid glands showed follicular heterogeneity with macrofollicles and numerous microfollicles without colloid, lined by flattened epithelial cells. In these thyroid follicles, there was intense TNFα (p = 0.002) staining, slight IL-6 staining (p = 0.042), and significantly stronger staining for IL-10 (p = 0.013) compared to that in the control group. This group also had a significantly lower body weight than the control animals on the 6th, 13th, and 20th days of life. In the SS2 group, the thyroids presented an architecture dominated by microfollicles without colloid as well as inflammatory cells in the colloid of some follicles. Immunohistochemistry revealed intense pan-follicular TNFα (p = 0.002) staining, with additional cytoplasmic staining of IL-6 (p = 0.040) and IL-10 (p = 0.006). The SS2 group also showed a more pronounced deficit compared to the SS1 group in terms of birth weight. The cranial–caudal length was shorter on the 13th and 20th days of life in the SS1 and SS2 groups compared to the control group. Conclusions: The results indicate that proximity of rats to the CPC was a determining factor in the development of histological abnormalities and increases in inflammatory cytokine markers in the thyroid glands of the offspring. In addition, the offspring born near the CPC had lower birth weights and shorter craniocaudal lengths compared to the animals in the control group. Full article

Hypoprolactinemia (hypoPRL) disrupts lactation and compromises milk production. Although maternal milk is a critical source of nutrients and bioactive compounds for newborns, the consequences of deficient lactation based on reduced milk quantity on the offspring’s immune development remain incompletely understood. Therefore, this study aimed to elucidate how deficient lactation due to hypoPRL interferes with offspring immunity and development. Female Sprague Dawley (SD) and spontaneous hypoPRL Oncins France Colony A (OFA) rats were euthanized on day 2 of lactation to assess the impact of hypoPRL on serum, milk, and tissue samples. We demonstrated that early deficient lactation in the OFA model impaired maternal performance, leading to increased pup mortality during early lactation. OFA pups exhibited reduced body weight and weight gain, decreased cerebral weight and index, and an increased cephalization index. Thymic development was markedly altered, as evidenced by reduced thymus weight, area, and cortical extension. These structural changes were accompanied by increased thymic Rag 1 expression, suggesting altered lymphocyte development. In parallel, passive immune transfer was compromised, with reduced levels of OVA-specific immunoglobulin isotypes detected in pup serum, reflecting changes in milk-derived immune support. In summary, maternal hypoPRL during early lactation adversely affects offspring growth and thymic maturation with lasting consequences for neonatal immune maturation. This study provides a novel experimental framework to investigate the consequences of lactation deficiency with potential implications for understanding lactation insufficiency and its impact on neonatal immune exposure. Full article

Apelin is a postnatal peptide implicated in gastrointestinal maturation, yet its combined effects on mucosa, enteric plexuses, and gut-derived appetite signals are not well defined. We investigated the impact of chronic intragastric apelin-13 on the stomach and small intestine of unweaned rats. Twelve Wistar pups of both sexes received apelin-13 (100 nmol/kg body weight, twice daily) or saline from postnatal day 10 for 14 days. After euthanasia, gastric and small-intestinal samples were processed for histomorphometry, neurofilament immunohistochemistry of myenteric and submucosal plexuses, and quantitative staining for ghrelin and leptin. Apelin-13 increased gastric mucosal thickness and pit and gland height, enlarged zymogen cells, and reduced muscularis propria thickness, while leaving submucosa and parietal cell area unchanged. In the small intestine, apelin produced a clear proximal-distal gradient, with enhanced villus-mucosa indices proximally and reduced indices in mid-to-distal jejunum, alongside broader crypt remodeling. Enterocyte and goblet cell dimensions changed in parallel with these regional shifts. Myenteric and submucosal ganglia were also remodeled in a segment-dependent manner. Ghrelin immunoreactivity increased in most regions, whereas leptin showed opposite proximal and distal responses. Overall, early-life luminal apelin-13 reshapes gastric and intestinal architecture and local hormone expression. Full article

The Zinc Finger X-Linked Duplicate B (ZXDB) gene is one of a pair of replicated zinc finger genes on chromosome Xp11.21. The homologous gene of ZXDB in mice is Zxdb. Recent studies have found that Zxdb plays a role in the spermatogenic process of mice; however, its impact on the female reproductive system has not yet been explored. In our study, we found, for the first time, that the loss of function of Zxdb leads to reduced decidualization rates and a decrease in litter size in female mice. Secondly, we found that maternal loss of Zxdb is the determinant of these phenotypes. Thirdly, the transcriptional and proteomic differential expression genes in the uterine tissues of wild-type (WT) and Zxdb knockout (Zxdb-KO) mice were significantly enriched in signaling pathways such as adhesion molecules. Finally, we demonstrated that the disorder of expression and uneven distribution of adhesion molecules in mouse uterine tissue may be the main reason for the decline in embryo implantation rate. In conclusion, we have established for the first time a link between the Zxdb gene and reduced female fertility. This study will help provide guidance and genetic counseling for future common clinical complications such as Recurrent Spontaneous Abortion (RSA) or Recurrent Implantation Failure (RIF). Full article

Molecular sex determination in Syrian hamsters (Mesocricetus auratus) has been limited by the incomplete annotation of Y-linked loci in currently available genome assemblies. Here, we evaluate the Y-linked gene PRSSLY, which encodes a testis-specific serine protease-like protein, as a molecular marker for genetic sexing of Syrian hamster embryonic and placental tissues. Primers flanking a conserved PRSSLY coding region produced a male-specific amplicon showing 100% concordance with results from the established KDM5C/KDM5D PCR assay in E15.5 tail biopsies. SYBR Green–based qPCR enables the accurate detection of PRSSLY, characterized by a unique melt-curve profile, exclusively in male samples, allowing for efficient and sensitive mid-throughput analysis. Application of the PRSSLY assay to 417 placental samples from 39 dams demonstrated its suitability for large-scale sex genotyping, enabling sex assignment in the majority of samples despite the intrinsic complexity of placental tissue containing both maternal and embryonic genetic material. This assay provides a robust and reproducible approach for accurate sex genotyping in developmental and reproductive studies using Syrian hamsters. Full article