A "toxic window" study on the hippocampal development of mice offspring exposed to azithromycin at different doses, courses, and time during pregnancy.

BACKGROUND: Azithromycin, one of the new-generation macrolides, is an effective medicine for the treatment of mycoplasma infection during pregnancy. Epidemiological studies have reported adverse pregnancy outcomes with prenatal azithromycin exposure (PAzE). However, the effect of PAzE on fetal hippocampal development is unclear. This study aimed to explore the effects and potential mechanism of PAzE-induced fetal hippocampal development at different doses, courses, and time. METHOD: Pregnant mice were administered azithromycin by gavage at different doses (50, 100 or 200 mg/kg.d), different courses (gestational day (GD)15-17 for three consecutive days, or GD17 once a day) and different time (GD10-12, GD15-17). RESULTS: Compared with the control group, morphological development damage of the fetal hippocampus was observed in the PAzE group, with a dysbalance in neuronal proliferation and apoptosis, decreased expression of the neuronal-specific marker Snap25, NeuN, PSD95 and Map2, increased expression of the glial-specific marker Iba1, GFAP, and S-100ß, and decreased expression of P2ry12. The PAzE-induced hippocampal developmental deficiency varied based on different doses, courses, and time, and the developmental toxicity was most significant in the late pregnancy, high dose, multi-course group (AZHT). The significant reduction of SOX2 and Wnt, which were related to regulation of neural progenitor cells (NPCs) proliferation in PAzE fetus compared with the control group indicated that the SOX2/Wnt signaling may be involved in PAzE-induced hippocampal developmental toxicity. CONCLUSION: In this study, PAzE was associated with hippocampal developmental toxicity in a variety of nerve cells. Hippocampal developmental toxicity due to azithromycin was most significant in the late pregnancy, high-dose (equivalent to maximum clinical dose) and multi-course group (AZHT). The findings provide an experimental and theoretical foundation for guiding the sensible use of medications during pregnancy and effectively assessing the risk of fetal hippocampal developmental toxicity.

Prenatal caffeine exposure induces autism-like behaviors in offspring under a high-fat diet via the gut microbiota-IL-17A-brain axis.

Prenatal caffeine exposure (PCE) is a significant contributor to intrauterine growth retardation (IUGR) in offspring, which has been linked to an increased susceptibility to autism spectrum disorder (ASD) later in life. Additionally, a high-fat diet (HFD) has been shown to exacerbate ASD-like behaviors, but the underlying mechanisms remain unclear. In this study, we first noted in the rat model of IUGR induced by PCE that male PCE offspring exhibited typical ASD-like behaviors post-birth, in contrast to their female counterparts. The female PCE offspring demonstrated only reduced abilities in free exploration and spatial memory. Importantly, both male and female PCE offspring displayed ASD-like behaviors when exposed to HFD. We further observed that PCE + HFD offspring exhibited damaged intestinal mucus barriers and disturbed gut microbiota, resulting in an increased abundance of Escherichia coli (E. coli). The induced differentiation of colonic Th17 cells by E. coli led to an increased secretion of IL-17A, which entered the hippocampus through peripheral circulation and caused synaptic damage in hippocampal neurons, ultimately resulting in ASD development. Our strain transplantation experiment suggested that E. coli-mediated increase of IL-17A may be the core mechanism of ASD with a fetal origin. In conclusion, PCE and HFD are potential risk factors for ASD, and E. coli-mediated IL-17A may play a crucial role in fetal-originated ASD through the gut-brain axis.

Microbiota-indole 3-propionic acid-brain axis mediates abnormal synaptic pruning of hippocampal microglia and susceptibility to ASD in IUGR offspring.

BACKGROUND: Autism spectrum disorder (ASD) has been associated with intrauterine growth restriction (IUGR), but the underlying mechanisms are unclear. RESULTS: We found that the IUGR rat model induced by prenatal caffeine exposure (PCE) showed ASD-like symptoms, accompanied by altered gut microbiota and reduced production of indole 3-propionic acid (IPA), a microbiota-specific metabolite and a ligand of aryl hydrocarbon receptor (AHR). IUGR children also had a reduced serum IPA level consistent with the animal model. We demonstrated that the dysregulated IPA/AHR/NF-κB signaling caused by disturbed gut microbiota mediated the hippocampal microglia hyperactivation and neuronal synapse over-pruning in the PCE-induced IUGR rats. Moreover, postnatal IPA supplementation restored the ASD-like symptoms and the underlying hippocampal lesions in the IUGR rats. CONCLUSIONS: This study suggests that the microbiota-IPA-brain axis regulates ASD susceptibility in PCE-induced IUGR offspring, and supplementation of microbiota-derived IPA might be a promising interventional strategy for ASD with a fetal origin. Video Abstract.

Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects.

The formation of learning and memory is regulated by synaptic plasticity in hippocampal neurons. Here we explored how gestational exposure to dexamethasone, a synthetic glucocorticoid commonly used in clinical practice, has lasting effects on offspring's learning and memory. Adult offspring rats of prenatal dexamethasone exposure (PDE) displayed significant impairments in novelty recognition and spatial learning memory, with some phenotypes maintained transgenerationally. PDE impaired synaptic transmission of hippocampal excitatory neurons in offspring of F1 to F3 generations, and abnormalities of neurotransmitters and receptors would impair synaptic plasticity and lead to impaired learning and memory, but these changes failed to carry over to offspring of F5 and F7 generations. Mechanistically, altered hippocampal miR-133a-3p-SIRT1-CDK5-NR2B signaling axis in PDE multigeneration caused inhibition of excitatory synaptic transmission, which might be related to oocyte-specific high expression and transmission of miR-133a-3p. Together, PDE affects hippocampal excitatory synaptic transmission, with lasting consequences across generations, and CDK5 in offspring's peripheral blood might be used as an early-warning marker for fetal-originated learning and memory impairment.

Impact of Prenatal Acetaminophen Exposure for Hippocampal Development Disorder on Mice.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used as analgesic agents. They have been detected in various environmental matrices. The degradation of environmental contaminants and the long-term adverse effects have become a major public concern. Prenatal exposure to acetaminophen can cause damage to the developing hippocampus. However, the molecular mechanisms behind hippocampal damage following prenatal acetaminophen exposure (PAcE) remain unclear. The present study shows an increased risk of adverse neurodevelopmental outcomes in offspring following exposure to acetaminophen during pregnancy on mice. The results revealed that different doses, timings, and duration of exposure to acetaminophen during pregnancy were associated with dose-dependent changes in the hippocampus of the offspring. Furthermore, exposure to high doses, multiple-treatment courses, and late pregnancy induced pathological changes, such as wrinkling and vacuolation, inhibited hippocampal proliferation and increased apoptosis. In addition, PAcE significantly decreased the expression of genes related to synaptic development in fetal hippocampal neurons and hippocampal astrocyte and microglia were also damaged to varying degrees. The significant reduction either in SOX2, an essential gene in regulating neural progenitor cell proliferation, and reduction of genes related to the SOX2/Notch pathway may suggest that the role of SOX2/Notch pathway in impaired hippocampal development in the offspring due to PAcE. In general, PAcE at high doses, multiple-treatment courses, and mid- and late gestation were associated with neurodevelopmental toxicity to the offspring.

MiR-17-5p/FOXL2/CDKN1B signal programming in oocytes mediates transgenerational inheritance of diminished ovarian reserve in female offspring rats induced by prenatal dexamethasone exposure.

Prenatal dexamethasone exposure (PDE) induces long-term reproductive toxicity in female offspring. We sought to explore the transgenerational inheritance effects of PDE on diminished ovarian reserve (DOR) in female offspring. Dexamethasone was subcutaneously administered into pregnant Wistar rats from gestational day 9 (GD9) to GD20 to obtain fetal and adult offspring of the F1 generation. F1 adult females were mated with normal males to produce the F2 generation, and the F3 generation. The findings showed decrease of serum levels of anti-Müllerian hormone (AMH) that in the PDE group, decrease in number of primordial follicles, and upregulation of miR-17-5p expression before birth in F1 offspring rats. Expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) and Forkhead Box L2 (FOXL2) were downregulated, and binding of FOXL2 and the CDKN1B promoter region was decreased in PDE groups of the F1, F2, and F3 generations. In vitro intervention experiments showed that glucocorticoid receptor (GR) was involved in activity of dexamethasone. These findings indicate that PDE can activate GR in fetal rat ovary and induce DOR of offspring, and its heritability is mediated by the cascade effect of miR-17-5p/FOXL2/CDKN1B. Increase in miR-17-5p expression in oocytes is the potential molecular basis for transgenerational inheritance of PDE effects.

Impact of prenatal amoxicillin exposure on hippocampal development deficiency.

BACKGROUND: Amoxicillin has been widely used to treat infectious diseases during pregnancy. Current studies suggest that amoxicillin exposure during pregnancy could lead to developmental disorders in the offspring and increase the incidence of long-term complications such as asthma and kidney damage in adulthood. However, the adverse effects of prenatal amoxicillin exposure (PAmE) including administration stage, doses and courses on fetal hippocampal neurodevelopment and its function in the offspring have not been elucidated. In this study, we intend to investigate the effects of PAmE on fetal hippocampal development and its possible mechanisms. METHOD: Pregnant Kunming mice were given intragastric administration with amoxicillin at different administration stage, doses and courses, and GD (gestational day) 18 offspring hippocampus was collected for morphological and development-related functional assays, and the molecular mechanisms were explored. RESULTS: PAmE induced hippocampal hypoplasia in the offspring with suppressed hippocampal neuronal cell proliferation and impaired neuronal synaptic plasticity comparatively; hippocampal astrocyte and microglia were damaged to varying degrees. The developmental toxicity of PAmE in fetal mices varies by time, dose, and course of treatment. The most severe damage was observed in the late gestation, high dose, and multi-course dosing groups. The significant reduction either in SOX2, an essential gene in regulating neural progenitor cell proliferation, and reduction of genes related to the Wnt/ß-catenin pathway may suggest that the key role of SOX2/Wnt/ß-catenin pathway in impaired hippocampal development in the offspring due to PAmE. CONCLUSION: In this study, PAmE was found to be developmentally toxic to the hippocampus thus to induce developmental damage to various hippocampal cells; Even with current clinically safe doses, potential hippocampal damage to offspring may still present; This study provides a theoretical and experimental basis for guiding the rational usage of drugs during pregnancy and giving effectively assessment of the risk on fetal hippocampal developmental toxicity.

Altered hippocampal GR/KCC2 signaling mediates susceptibility to convulsion in male offspring following dexamethasone exposure during pregnancy in rats.

Epidemiological research suggests that convulsions may have an intrauterine developmental origin related to the application of dexamethasone, an artificially synthesized glucocorticoid. Here, using a rat animal model of prenatal dexamethasone exposure (PDE) we confirm that PDE can cause susceptibility to convulsions in male offspring and explore the epigenetic programming mechanism underlying this effect related to intrauterine type 2 K+-Cl- cotransporter (KCC2). Wistar rats were injected with dexamethasone (0.2 mg/kg/d) subcutaneously during the gestational days (GD) 9-20 and part of the offspring was given lithium pilocarpine (LiPC) at postnatal week 10. Our results showed that male offspring of the PDE+LiPC group exhibited convulsions susceptibility, as well as increased hippocampal gamma-aminobutyric acid (GABA) and intracellular chloride ions level and decreased GABA receptor expression. The offspring also showed a decrease of hippocampal KCC2 H3K14ac levels and KCC2 expression. PDE male fetal rats (GD20) showed similar changes to male offspring after birth and exhibited an increased expression of glucocorticoid receptor (GR) and histone deacetylase type 2 (HDAC2). We observed effects consistent with those observed in PDE fetal rats following in vitro dexamethasone treatment of the fetal rat hippocampal neuron H19-7 cell line, and the effects could be reversed by treatment with a GR inhibitor (RU486) or HDAC2 inhibitor (romidepsin). Taken together, this study confirmed that PDE causes a reduction of H3K14ac levels in the KCC2 promoter region caused by activation of fetal hippocampal GR-HDAC2-KCC2 signaling. We proposed that this abnormal epigenetic modification is the mechanism underlying offspring convulsions susceptibility. CATEGORIES: Mechanism of toxicity.

Programming changes of hippocampal miR-134-5p/SOX2 signal mediate the susceptibility to depression in prenatal dexamethasone-exposed female offspring.

Depression is a neuropsychiatric disorder and has intrauterine developmental origins. This study aimed to confirm the depression susceptibility in offspring rats induced by prenatal dexamethasone exposure (PDE) and to further explore the intrauterine programming mechanism. Wistar rats were injected with dexamethasone (0.2 mg/kg·d) subcutaneously during the gestational days 9-20 and part of the offspring was given chronic stress at postnatal weeks 10-12. Behavioral results showed that the adult PDE female offspring was susceptible to depression, accompanied by increased hippocampal miR-134-5p expression and decreased sex-determining region Y-box 2 (SOX2) expression, as well as disorders of neural progenitor cells proliferation and hippocampal neurogenesis. The PDE female fetal rats presented consistent changes with the adult offspring, accompanied by the upregulation of glucocorticoid receptor (GR) expression and decreased sirtuin 1 (SIRT1) expression. We further found that the H3K9ac level of the miR-134-5p promoter was significantly increased in the PDE fetal hippocampus, as well as in adult offspring before and after chronic stress. In vitro, the changes of GR/SIRT1/miR-134-5p/SOX2 signal by dexamethasone were consistent with in vivo experiments, which could be reversed by GR receptor antagonist, SIRT1 agonist, and miR-134-5p inhibitor. This study confirmed that PDE led to an increased expression level as well as H3K9ac level of miR-134-5p by activating the GR/SIRT1 pathway in the fetal hippocampus and then inhibited the SOX2 expression. The programming effect mediated by the abnormal epigenetic modification could last from intrauterine to adulthood, which constitutes the intrauterine programming mechanism leading to hippocampal neurogenesis disorders and depression susceptibility in female offspring. Intrauterine programming mechanism for the increased depressive susceptibility in adult female offspring by prenatal dexamethasone exposure (PDE). GR, glucocorticoid receptor; SIRT1, sirtuin 1; SOX2, sex-determining region Y-box 2; NPCs, neuroprogenitor cells; H3K9ac, histone 3 lysine 9 acetylation; GRE, glucocorticoid response element.

The combination of hprt and gapdh is the best compound reference genes in the fetal rat hippocampus.

Hippocampus, as an important organ of central memory storage and spatial orientation, has been studied increasingly in recent years. The expression of reference genes in the hippocampus of adult rats, which are commonly used in the quantitative real-time polymerase chain reaction (qRT-PCR), is unstable in the fetal hippocampus and may not be suitable for the fetal period. Therefore, this study intends to screen and determine the optimal compound reference genes in the fetal rat hippocampus. Based on the literature, we selected five housekeeping genes (HKGs), including glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin beta (ß-actin), hypoxanthine phosphoribosyltransferase (hprt), 18s ribosomal RNA (18s rRNA), and cyclophilin B (cypB). We analyzed the expression of them under physiological conditions in the fetal rat hippocampus using BestKeeper, GeNorm, and NormFinder, to select the most stable compound reference genes. Furthermore, to verify the stability of the compound reference genes, we analyzed the expression of reference genes in the fetal rat hippocampus under the pathological model of prenatal dexamethasone exposure (PDE). Finally, we evaluated the accuracy of compound reference genes through detecting the expression of fetal rat hippocampal brain-derived neurotrophic factor (BDNF) under PDE model. This study determined that the combination of gapdh and hprt was the most stable and suitable compound reference genes in the fetal rat hippocampus. There was no significant difference between male and female fetal rats. We provided the support of accurate and reliable reference genes for the further study of diseases related to the fetal hippocampus.