Embryo Brain Research Articles

Brain Specific RagA Overexpression Triggers Depressive-Like Behaviors in Mice via Activating ADORA2A Signaling Pathway.

Neuroinflammation hallmarks the pathology of depression although the etiological complexity has not yet been resolved. Previous studies demonstrate that bacterial lipopolysaccharide induces depressive-like behaviors by activating RagA-mTOR-p70S6K signaling pathway. The current project aims to investigate whether and how brain-specific RagA overexpression triggers depressive-like behaviors in mice. Full-length RagA cDNA is cloned into the mammalian expression vector under the control of brain specific promoter, and subsequently overexpressed in the brain of mouse embryos. Indeed, RagA transgenic mice exhibit depressive-like behaviors and memory impairments. RNA-seq profiling of the prefrontal cortex (PFC) transcriptome highlights adenosine A2a receptor (ADORA2A) as a key differentially expressed gene (DEG). Western blotting confirms that ADORA2A and phospho-p70S6K are markedly elevated in RagA transgenic mice. Behavioral assessments demonstrate that ADORA2A inhibitor istradefylline markedly attenuates depressive-like behaviors. Further metabolomics reveals that N-acetylserotonin and several depression-related metabolites are downregulated while proteomic profiling showed that OLIG1 and other proteins are significantly regulated in RagA transgenic mice. Collectively, RagA overexpression alters the expression patterns of signaling proteins and the metabolism of depression-associated metabolites. RagA may cause depressive-like behaviors in mice via activating p70S6K/ADORA2A signaling pathway. Thus, RagA-p70S6K-ADORA2A signaling pathway may be a target for the development of new antidepressant therapies.

The Impact of Mobile Phone Electromagnetic Waves on the Neurons and Blood Brain Barrier Integrity in the Chick Embryo

The electromagnetic environment surrounding us has dramatically evolved over the past decade, with the proliferation of Wi-Fi, Bluetooth, and other wireless technologies becoming commonplace in our daily lives. Mobile phones emit non-ionizing low-frequency electromagnetic waves (EW). To examine the effects of EW on living cells, this study aims to explore the impact of cell phone EW on the developing brain of chick embryos. The fertilized eggs were allowed to develop under exposure to electromagnetic waves emitted by cell mobile. A cell phone was placed inside the incubator with 20 eggs and was called from outside on a precise schedule. The same number of fertilized eggs were placed in another incubator without a mobile phone and served as the control. Embryos were sacrificed on days 10 and 15, and the cerebral cortex and cerebellum were removed and sent for electron microscopy. In the control group, cerebral neurons appeared healthy, with a large, centrally placed nucleus, visible oligodendrocytes, and a less dense extracellular matrix. In contrast, neurons from the exposed group were smaller, fewer in number, with unclear nuclear margins, signs of shrinkage, and apoptosis and a dense extracellular matrix. In the cerebellum, the exposed group revealed a reduced number of Purkinje neurons and noticeable mitochondrial swelling. The blood-brain barrier remained intact in the control group but was compromised in the exposed group. We conclude that electromagnetic waves emitted by cell phones adversely affect the normal development of the brain in chick embryos.

Large field of view and spatial region of interest transcriptomics in fixed tissue

Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and formalin fixation and paraffin embedding (FFPE) tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis to promote the development of transcriptomics.

IER3IP1-mutations cause microcephaly by selective inhibition of ER-Golgi transport

Mutations in the IER3IP1 (Immediate Early Response-3 Interacting Protein 1) gene can give rise to MEDS1 (Microcephaly with Simplified Gyral Pattern, Epilepsy, and Permanent Neonatal Diabetes Syndrome-1), a severe condition leading to early childhood mortality. The small endoplasmic reticulum (ER)-membrane protein IER3IP1 plays a non-essential role in ER-Golgi transport. Here, we employed secretome and cell-surface proteomics to demonstrate that the absence of IER3IP1 results in the mistrafficking of proteins crucial for neuronal development and survival, including FGFR3, UNC5B and SEMA4D. This phenomenon correlates with the distension of ER membranes and increased lysosomal activity. Notably, the trafficking of cargo receptor ERGIC53 and KDEL-receptor 2 are compromised, with the latter leading to the anomalous secretion of ER-localized chaperones. Our investigation extended to in-utero knock-down of Ier3ip1 in mouse embryo brains, revealing a morphological phenotype in newborn neurons. In summary, our findings provide insights into how the loss or mutation of a 10 kDa small ER-membrane protein can cause a fatal syndrome.

Altered Morpho-Functional Features of Neurogenesis in Zebrafish Embryos Exposed to Non-Combustion-Derived Magnetite.

Neurogenesis is the process by which new brain cells are formed. This crucial event emerges during embryonic life and proceeds in adulthood, and it could be influenced by environmental pollution. Non-combustion-derived magnetite represents a portion of the coarse particulate matter (PM) contributing to air and water pollution in urban settings. Studies on humans have reported that magnetite and other iron oxides have significant damaging effects at a central level, where these particles accumulate and promote oxidative stress. Similarly, magnetite nanoparticles can cross the placenta and damage the embryo brain during development, but the impact on neurogenesis is still unknown. Furthermore, an abnormal Fe cation concentration in cells and tissues might promote reactive oxygen species (ROS) generation and has been associated with multiple neurodegenerative conditions. In the present study, we used zebrafish as an in vivo system to analyze the specific effects of magnetite on embryonic neurogenesis. First, we characterized magnetite using mineralogical and spectroscopic analyses. Embryos treated with magnetite at sub-lethal concentrations showed a dose-response increase in ROS in the brain, which was accompanied by a massive decrease in antioxidant genes (sod2, cat, gsr, and nrf2). In addition, a higher number of apoptotic cells was observed in embryos treated with magnetite. Next, interestingly, embryos exposed to magnetite displayed a decrease in neural staminal progenitors (nestin, sox2, and pcna markers) and a neuronal marker (elavl3). Finally, we observed significative increases in apoeb (specific microglia marker) and interleukin-1b (il1b), confirming a status of inflammation in the brain embryos treated with magnetite. Our study represents the very first in vivo evidence concerning the effects of magnetite on brain development.

Maternal obesity alters fetal neuroinflammation in a murine model of preterm birth

BackgroundPreterm birth from intrauterine infection is a leading cause of neonatal neurologic morbidity. Maternal obesity is also associated with intra-amniotic infection and inflammation. It remains unknown if maternal obesity is a risk factor for fetal brain injury that occurs with premature birth. We hypothesize that maternal obesity intensifies fetal neuroinflammation within the setting of premature delivery. ObjectiveUtilizing a murine model, we aimed to examine the influence of maternal obesity on perinatal neuroinflammatory responses that arise with preterm birth. Study DesignObese dams were generated via a high-fat diet that was maintained through pregnancy. In parallel, non-obese (normal) dams received a control diet. All dams were paired with males on normal diet. Pregnant dams were randomized to receive an intrauterine injection of bacterial endotoxin (LPS) or the vehicle (PBS) on embryo day 15.5 of what it typically a 19-21 day gestation. Fetal brains were harvested 6 hours following injections and the expression of key inflammatory cytokines (Il1b, Il6, and Tnf) as well as panels of metabolic, immune, and inflammatory genes was analyzed. ResultsWith PBS there were no differences in gene expression related to maternal obesity. There were substantial differences in Il6 and immune/inflammatory expression profiles in fetal brains from obese versus normal dams that received LPS. Few differences were observed among the metabolic genes examined under these conditions. Gene expression patterns with maternal obesity associated with pathways related to white matter injury. ConclusionThe expression of neuroinflammatory markers instigated by bacterial endotoxin via intrauterine LPS, was greater in embryo brains obtained from obese dams. Expression profiles suggest that in combination with intrauterine inflammation, maternal obesity may increase the risk of fetal white matter injury. Further investigation is warranted towards understanding the relationship between maternal health and neurologic outcomes associated with prematurity.

Epigallocatechin-3-gallate-synthesized gold nanoparticles modulate reactive oxygen species and antioxidant parameters in brain and heart tissues using a chicken embryo model

Chemically synthesized gold nanoparticles (AuNPs) come with drawbacks, such as biological toxicity and ecological imbalances. Nonetheless, a knowledge gap remains regarding their impact on oxidative and antioxidant parameters. Here, environmentally friendly AuNPs were synthesized using epigallocatechin-3-gallate (AuNPs-EGCG). The successful synthesis of AuNPs-EGCG were confirmed using ultraviolet–visible spectroscopy, Fourier-transform infrared spectroscopy, atomic force microscopy, and dynamic light scattering. Antioxidant activity was assessed using free radical 2,2-diphenyl-1-picrylhydrazyl, oxygen radical absorbance capacity, and ferric reducing antioxidant power assays, while cytotoxicity properties were evaluated in the NIH3T3 fibroblast cells. Furthermore, the impact of AuNPs-EGCG on oxidative stress was investigated in the hearts and brains of chicken embryos. AuNPs-EGCG exhibited no cytotoxicity and was not toxic for chicken embryos at higher concentrations. However, exposure to lower concentrations (100 and 30 μg/mL) caused an increase in reactive oxygen species (ROS) levels and a decrease in total antioxidant capacity in the heart and brain, resulting in a disturbance in the redox balance attributed to a reduction in catalase activity and glutathione (GSH) content, and an increase in glutathione peroxidase activity. In contrast, AuNPs-EGCG at 300 μg/mL promoted an increase in GSH levels and modulation of ROS, while also reducing vascular density in the chorioallantoic membrane. Furthermore, AuNPs-EGCG did not cause lipid or protein oxidation. The present study offers evidence that supports the investigation of how AuNPs-EGCG can modulate antioxidant defense mechanisms and assess the safety of phytoantioxidant-functionalized nanoparticles in vivo. This may lead to new and innovative strategies for the utilization of nanomaterials in biological systems.

Establishment of a new equine embryo brain primary cell culture with long-term expansion

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Lumen Pressure Modulation in Chicken Embryos.

Pressure exerted by fluid contained within a lumen plays a crucial role in the growth, morphogenesis, and patterning of epithelial organs. Accurate modulation of lumen pressure in the developing embryo requires sensitive and robust methods that can detect and vary pressure in the range of tens to hundreds of Pascals (Pa). Here we describe a simple, cost-effective protocol for setting up a pressure modulation apparatus combining a high-sensitivity pressure sensor and a water column whose height can be finely tuned. We demonstrate lumen pressure control using the developing brain of early chicken embryos.

A simple method for preservation of embryonic rat brain tissue for primary neuron culture

Aims/Purpose: In this study we aim to investigate the effect of short‐term storage of embryonic rat brain tissue in Hibernate‐E medium to be used in the isolation of primary neurons for in vitro biocompatibility and electrophysiology tests of retinal and optic nerve prosthesis. Functionally viable neuron cells will be an alternative method for regular dissection from tissue for further experiments.Methods: Hippocampal tissues from E15‐E17 embryos of Wistar Albino rat were dissecting for primary neuron culture. Half of the tissues were isolated on the same day and used as a control group for further experiments. The test group was stored in a Hibernate‐E medium for 1 week and the same isolation method was performed for the test group. For tissue preservation, half medium replacement with fresh Hibernate‐E medium on the 4th day was enhanced cell viability. Morphology, characterization and synapse formation were determined with immunoflorescence staining.Results: Comparison of neuronal survival between cultures prepared from stored hippocampi and freshly isolated hippocampi resulted in similar neuronal survival rates in both preparations with cell viability assays. Cell viability rate of hippocampus tissue isolated from rat embryo brain was demonstrated as 96% and cell viability rate of preserved tissue in Hibernate‐E Medium for 7 days was revealed as 90.22% by trypan blue cell viability test (n = 4). Morphological and immunocytochemical characterizations of stored and fresh hippocampal cultures were similar by observing beta‐III Tubulin neuron cytoskeleton biomarker.Conclusions: As a conclusion, our method has the potential to increase repeatability of primary neuron cultures within 1 week and reduce the number of isolation steps in research. In addition, it will provide a great convenience in the experimental timelines of the researchers and contribute to the reduction of the workload despite any obstacles during primary neuron culture.

Selenomethionine alleviates decabromodiphenyl ether-induced oxidative stress and ferroptosis via the NRF2/GPX4 pathway in the chicken brain

Decabromodiphenyl ether (BDE209) is a toxic environmental pollutant that can cause neurotoxicity, behavioral abnormalities, and cognitive impairment in animals. However, the specific mechanisms of BDE209-induced neurological injury and effective preventative and therapeutic interventions are lacking. Even though selenomethionine (Se-Met) has a significant detoxification effect and protects the nervous system, it remains unclear whether Se-Met can counteract the toxic effects of BDE209. For the in vivo test, we randomly divided 60 1-week-old hy-line white variety chicks into the Con, BDE209, Se-Met, and BDE209 +Se-Met groups. In vitro experiments were performed, exposing chick embryo brain neurons to BDE209, Se-Met, N-Acetylcysteine (NAC, a ROS inhibitor), and RSL3 (a GPX4 inhibitor). We demonstrated that BDE209 induced oxidative stress and ferroptosis in the chicken brain, which mainly manifested as mitochondrial atrophy, cristae breakage, increased Fe2+ and MDA content, decreased antioxidant enzyme activity, and the inhibition of the NRF2/GPX4 signaling pathway in the brain neurons. However, Se-Met supplementation reversed these changes by activating the NRF2/GPX4 pathway, reducing mitochondrial damage, enhancing antioxidant enzyme activity, and alleviating ferroptosis. This study provides insight into the mechanism of BDE209-related neurotoxicity and suggests Se-Met as an effective preventative and control measure against BDE209 poisoning.

The developing brain in the formation of oxidant and antioxidant systems

BACKGROUND: The structures, tissues, and systems of the brain differentiate gradually. The values of biochemical constants vary depending on the timing of development, that is, the embryonic, early, and late postnatal periods. In this respect, the multicomponent oxidation and antioxidation systems that do not mature at the same time are of interest.
 AIM: To examine the processes of lipid peroxidation based on the level of malonic dialdehyde and antioxidant protection (superoxide dismutase, catalase, and reduced glutathione) in the brain of rat embryos and offspring at different periods of pre- and postnatal development.
 MATERIALS AND METHODS: Thirty-nine pregnant female Wistar rats weighing 220–250 g were selected, from which 176 embryos and rat pups of different sexes and age were obtained, including embryos in the third trimester of pregnancy (13–17 days of gestation) and rat pups aged 1–14 weeks. The concentration of malonic dialdehyde (indicator of lipid peroxidation) was determined in the brain tissue, and the activity of superoxide dismutase, catalase, and level of reduced glutathione was found as indicators of antioxidant defense systems.
 RESULTS: The brains of the embryos were characterized by low levels of malonic dialdehyde, and its concentration sharply increased immediately after the birth of rat pups. A similar but a less pronounced pattern was also recorded for indicators of antioxidant protection (superoxide dismutase activity and level of reduced glutathione). The opposite reaction was observed in catalase, which demonstrated high activity in the brain in the prenatal period but significantly decreased after birth. With further postnatal development up to sexual maturity (14 weeks, or 3 months of age), no significant changes in the activities of superoxide dismutase, catalase, and concentration of reduced glutathione were noted; however, a twofold drop in the level of malonic dialdehyde in the brain was noted.
 CONCLUSION:Already in the first months of life in rats, a quite stable status of lipid peroxidation and antioxidant defense systems of the brain tissue developed.

Long-acting dolutegravir formulations prevent neurodevelopmental impairments in a mouse model.

The World Health Organization has recommended dolutegravir (DTG) as a preferred first-line treatment for treatment naive and experienced people living with human immunodeficiency virus type one (PLWHIV). Based on these recommendations 15 million PLWHIV worldwide are expected to be treated with DTG regimens on or before 2025. This includes pregnant women. Current widespread use of DTG is linked to the drug's high potency, barrier to resistance, and cost-effectiveness. Despite such benefits, potential risks of DTG-linked fetal neurodevelopmental toxicity remain a concern. To this end, novel formulation strategies are urgently needed in order to maximize DTG's therapeutic potentials while limiting adverse events. In regard to potential maternal fetal toxicities, we hypothesized that injectable long-acting nanoformulated DTG (NDTG) could provide improved safety by reducing drug fetal exposures compared to orally administered native drug. To test this notion, we treated pregnant C3H/HeJ mice with daily oral native DTG at a human equivalent dosage (5mg/kg; n = 6) or vehicle (control; n = 8). These were compared against pregnant mice injected with intramuscular (IM) NDTG formulations given at 45 (n = 3) or 25 (n = 4) mg/kg at one or two doses, respectively. Treatment began at gestation day (GD) 0.5. Magnetic resonance imaging scanning of live dams at GD 17.5 was performed to obtain T1 maps of the embryo brain to assess T1 relaxation times of drug-induced oxidative stress. Significantly lower T1 values were noted in daily oral native DTG-treated mice, whereas comparative T1 values were noted between control and NDTG-treated mice. This data reflected prevention of DTG-induced oxidative stress when delivered as NDTG. Proteomic profiling of embryo brain tissues harvested at GD 17.5 demonstrated reductions in oxidative stress, mitochondrial impairments, and amelioration of impaired neurogenesis and synaptogenesis in NDTG-treated mice. Pharmacokinetic (PK) tests determined that both daily oral native DTG and parenteral NDTG achieved clinically equivalent therapeutic plasma DTG levels in dams (4,000-6,500ng/mL). Importantly, NDTG led to five-fold lower DTG concentrations in embryo brain tissues compared to daily oral administration. Altogether, our preliminary work suggests that long-acting drug delivery can limit DTG-linked neurodevelopmental deficits.

New insights into brain injury in chickens induced by bisphenol A and selenium deficiency—Mitochondrial reactive oxygen species and mitophagy-apoptosis crosstalk homeostasis

Bisphenol A (BPA), a component of plastic products, can penetrate the blood-brain barrier and pose a threat to the nervous system. Selenium (Se) deficiency can also cause nervous system damage. Resulting from the rapid industrial development, BPA pollution and Se deficiency often coexist. However, it is unclear whether brain damage in chickens caused by BPA exposure and Se deficiency is related to the crosstalk disorder between mitophagy and apoptosis. In this study, 60 chickens (1 day old) were fed with a diet that contained 20 mg/kg BPA but was insufficient in Se (only 0.039 mg/kg) for 42 days to establish a chicken brain injury model. In vitro, the primary chicken embryo brain neurons were treated for 24 h with Se-deficient medium containing 75 μM BPA. The results showed that BPA exposure and Se deficiency inhibited the expression of the mitochondrial respiratory chain complex in brain neurons, and a large number of mitochondrial reactive oxygen species were released. Furthermore, the expression levels of mitochondrial fusion proteins (OPA1, Mfn1, and Mfn2) decreased, while the expression levels of mitochondrial fission proteins (Drp1, Mff, and Fis1) increased, thus exacerbating mitochondrial division. In addition, the results of immunofluorescence and flow cytometry analysis, as well as the elevated expressions of mitophagy related genes (PINK1, Parkin, ATG5, and LC3II/I) and pro-apoptotic markers (Bax, Cytc, Caspase3, and Caspase9) indicated that BPA exposure and Se deficiency disrupted the crosstalk homeostasis between mitophagy and apoptosis. However, this crosstalk homeostasis was restored after Mito-Tempo and Rapamycin treatment. In contrast, 3-methyladenine treatment exacerbated this crosstalk disorder. In conclusion, BPA exposure and Se deficiency can induce mitochondrial reactive oxygen species bursts and disorders of mitochondrial dynamics by destroying the mitochondrial respiratory chain complex. The result is indicative of an imbalance in mitochondrial autophagy and apoptosis crosstalk homeostasis, which damages the chicken brain.

Urine Excretion, Organ Distribution, and Placental Transfer of 6PPD and 6PPD-Quinone in Mice and Potential Developmental Toxicity through Nuclear Receptor Pathways.

The rubber antioxidant 6PPD has gained significant attention due to its highly toxic transformation product, 6PPD-quinone (6PPDQ). Despite their detection in urines of pregnant women, the placental transfer and developmental toxicity of 6PPD and 6PPDQ are unknown. Here, we treated C57Bl/6 mice with 4 mg/kg 6PPD or 6PPDQ to investigate their urine excretion and placental transfer. Female and male mice exhibited sex difference in excretion profiles of 6PPD and 6PPDQ. Urine concentrations of 6PPDQ were one order of magnitude lower than those of 6PPD, suggesting lower excretion and higher bioaccumulation of 6PPDQ. In pregnant mice treated with 6PPD or 6PPDQ from embryonic day 11.5 to 15.5, 6PPDQ showed ∼1.5-8 times higher concentrations than 6PPD in placenta, embryo body, and embryo brain, suggesting higher placental transfer of 6PPDQ. Using in vitro dual-luciferase reporter assays, we revealed that 6PPDQ activated the human retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα) at concentrations as low as 0.3 μM, which was ∼10-fold higher than the concentrations detected in human urines. 6PPD activated the RXRα at concentrations as low as 1.2 μM. These results demonstrate the exposure risks of 6PPD and 6PPDQ during pregnancy and emphasize the need for further toxicological and epidemiological investigations.

Oxytetracycline changes the behavior of zebrafish larvae by inhibiting NMDA receptors

Oxytetracycline (OTC), a tetracycline antimicrobial, is one of the antimicrobial drugs frequently used in the aquaculture and livestock industries. Due to its extensive usage and emissions, OTC has been identified as a significant new emerging pollutant (EP) in a number of environments. OTC frequently causes toxic effects on the central nervous system, but it can be challenging to monitor, and it is still unclear how these toxicities are caused. We used bioinformatic analysis techniques to screen for OTC targets and discovered that NMDA receptors are potential targets of OTC neurotoxicity. To confirm this finding, we exposed zebrafish embryos to 5 mg/L OTC-containing rearing water from 2-hour post fertilization (hpf) to 8-day post fertilization (dpf), performed spontaneous movement and light-dark stimulation assays at 6 and 8 dfp, and discovered that OTC inhibited locomotor activity and attenuated anxiety-like responses in zebrafish larvae. Meanwhile, the qPCR and immunofluorescence staining results suggested that OTC inhibited the expression of multiple subtypes of NMDA receptors (grin1a, grin1b, grin2bb, grin2ca) and induced apoptosis in the brains of zebrafish embryos. Simultaneous administration of NMDA, an NMDA receptor agonist, completely antagonized the inhibitory neurobehavioral changes in zebrafish larvae, as well as the downregulation of N-methyl-D-aspartate (NMDA) receptor expression and apoptosis in the embryonic brains caused by OTC exposure. In conclusion, OTC exhibited significant inhibitory neurobehavioral toxicity in zebrafish larvae during early development, which may be dependent on its suppression of NMDA receptor activity and expression. Furthermore, OTC-induced neurodevelopmental toxicity may be associated with NMDA receptor-regulated neuronal apoptosis.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior.

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 µm) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 µm) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Short-Term Exposure to Benzo(a)Pyrene Causes Disruption of GnRH Network in Zebrafish Embryos.

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is considered a common endocrine disrupting chemical (EDC) with mutagenic and carcinogenic effects. In this work, we evaluated the effects of BaP on the hypothalamo-pituitary-gonadal axis (HPG) of zebrafish embryos. The embryos were treated with 5 and 50 nM BaP from 2.5 to 72 hours post-fertilization (hpf) and obtained data were compared with those from controls. We followed the entire development of gonadotropin releasing hormone (GnRH3) neurons that start to proliferate from the olfactory region at 36 hpf, migrate at 48 hpf and then reach the pre-optic area and the hypothalamus at 72 hpf. Interestingly, we observed a compromised neuronal architecture of the GnRH3 network after the administration of 5 and 50 nM BaP. Given the toxicity of this compound, we evaluated the expression of genes involved in antioxidant activity, oxidative DNA damage and apoptosis and we found an upregulation of these pathways. Consequently, we performed a TUNEL assay and we confirmed an increment of cell death in brain of embryos treated with BaP. In conclusion our data reveal that short-term exposure of zebrafish embryos to BaP affects GnRH3 development likely through a neurotoxic mechanism.

Polysarcosine-based lipid formulations for intracranial delivery of mRNA.

Messenger RNA (mRNA) is revolutionizing the future of therapeutics in a variety of diseases, including neurological disorders. Lipid formulations have shown to be an effective platform technology for mRNA delivery and are the basis for the approved mRNA vaccines. In many of these lipid formulations, polyethylene glycol (PEG)-functionalized lipid provides steric stabilization and thus plays a key role in improving the stability both ex vivo and in vivo. However, immune responses towards PEGylated lipids may compromise the use of those lipids in some applications (e.g., induction of antigen specific tolerance), or within sensitive tissues (e.g., central nervous system (CNS)). With respect to this issue, polysarcosine (pSar)-based lipopolymers were investigated as an alternative to PEG-lipid in mRNA lipoplexes for controlled intracerebral protein expression in this study. Four polysarcosine-lipids with defined sarcosine average molecular weight (Mn=2k, 5k) and anchor diacyl chain length (m=14, 18) were synthesized, and incorporated into cationic liposomes. We found that the content, pSar chain length and carbon tail lengths of pSar-lipids govern the transfection efficiency and biodistribution. Increasing carbon diacyl chain length of pSar-lipid led up to 4- and 6-fold lower protein expression in vitro. When the length of either pSar chain or lipid carbon tail increased, the transfection efficiency decreased while the circulation time was prolonged. mRNA lipoplexes containing 2.5% C14-pSar2k resulted in the highest mRNA translation in the brain of zebrafish embryos through intraventricular injection, while C18-pSar2k-liposomes showed a comparable circulation with DSPE-PEG2k-liposomes after systemic administration. To conclude, pSar-lipid enable efficient mRNA delivery, and can substitute PEG-lipids in lipid formulations for controlled protein expression within the CNS.