Maternal n-3 enriched diet reprograms the offspring neurovascular transcriptome and blunts inflammation induced by endotoxin in the neonate.

Infection during the perinatal period can adversely affect brain development, predispose infants to ischemic stroke and have lifelong consequences. We previously demonstrated that diet enriched in n-3 polyunsaturated fatty acids (n-3 PUFA) transforms brain lipid composition in the offspring and protects the neonatal brain from stroke, in part by blunting injurious immune responses. Critical to the interface between the brain and systemic circulation is the vasculature, endothelial cells in particular, that support brain homeostasis and provide a barrier to systemic infection. Here, we examined whether maternal PUFA-enriched diets exert reprograming of endothelial cell signalling in postnatal day 9 mice after modeling aspects of infection using LPS. Transcriptome analysis was performed on microvessels isolated from brains of pups from dams maintained on 3 different maternal diets from gestation day 1: standard, n-3 enriched or n-6 enriched diets. Depending on the diet, in endothelial cells LPS produced distinct regulation of pathways related to immune response, cell cycle, extracellular matrix, and angiogenesis. N-3 PUFA diet enabled higher immune reactivity in brain vasculature, while preventing imbalance of cell cycle regulation and extracellular matrix cascades that accompanied inflammatory response in standard diet. Cytokine analysis revealed a blunted LPS response in blood and brain of offspring from dams on n-3 enriched diet. Analysis of cerebral vasculature in offspring in vivo revealed no differences in vessel density. However, vessel complexity was decreased in response to LPS at 72 h in standard and n-6 diets. Thus, LPS modulates specific transcriptomic changes in brain vessels of offspring rather than major structural vessel characteristics during early life. N-3 PUFA-enriched maternal diet in part prevents an imbalance in homeostatic processes, alters inflammation and ultimately mitigates changes to the complexity of surface vessel networks that result from infection. Importantly, maternal diet may presage offspring neurovascular outcomes later in life.

Gut microbiota differences in five-year-old children that were born preterm with a history of necrotizing enterocolitis: A pilot trial.

The study explores the long-term effects of necrotizing enterocolitis (NEC) on gut microbiota in preterm infants by analyzing stool samples from 5-year-old children using shotgun metagenomic sequencing. It compares children with a history of NEC, treated surgically or medically, to preterm controls without NEC. Findings reveal persistent gut microbiota dysbiosis in NEC children, with reduced species diversity and evenness, especially in those treated surgically. The surgical NEC group had a lower Shannon index, indicating less microbial diversity. Significant differences in taxonomic profiles were observed, mainly influenced by surgical treatment. These results underscore the lasting impact of NEC and its treatment on gut microbiota, suggesting a need for strategies addressing long-term dysbiosis.

Approaches to embryonic neurodevelopment: from neural cell to neural tube formation through mathematical models.

The development of the human central nervous system initiates in the early embryonic period until long after delivery. It has been shown that several neurological and neuropsychiatric diseases originate from prenatal incidents. Mathematical models offer a direct way to understand neurodevelopmental processes better. Mathematical modelling of neurodevelopment during the embryonic period is challenging in terms of how to 'Approach', how to initiate modelling and how to propose the appropriate equations that fit the underlying dynamics of neurodevelopment during the embryonic period while including the variety of elements that are built-in naturally during the process of neurodevelopment. It is imperative to answer where and how to start modelling; in other words, what is the appropriate 'Approach'? Therefore, one objective of this study was to tackle the mathematical issue broadly from different aspects and approaches. The approaches were divided into three embryonic categories: cell division, neural tube growth and neural plate growth. We concluded that the neural plate growth approach provides a suitable platform for simulation of brain formation/neurodevelopment compared to cell division and neural tube growth. We devised a novel equation and designed algorithms that include geometrical and topological algorithms that could fit most of the necessary elements of the neurodevelopmental process during the embryonic period. Hence, the proposed equations and defined mathematical structure would be a platform to generate an artificial neural network that autonomously grows and develops.

Maternal n-3 enriched diet reprograms neurovascular transcriptome and blunts inflammation in neonate.

Infection during perinatal period can adversely affect brain development, predispose infants to ischemic stroke and have lifelong consequences. We previously demonstrated that diet enriched in n-3 polyunsaturated fatty acids (PUFA) transforms brain lipid composition and protects from neonatal stroke. Vasculature is a critical interface between blood and brain providing a barrier to systemic infection. Here we examined whether maternal PUFA-enriched diets exert reprograming of endothelial cell signalling in 9-day old mice after endotoxin (LPS)-induced infection. Transcriptome analysis was performed on brain microvessels from pups born to dams maintained on 3 diets: standard, n-3 or n-6 enriched. N-3 diet enabled higher immune reactivity in brain vasculature, while preventing imbalance of cell cycle regulation and extracellular matrix cascades that accompanied inflammatory response in standard diet. LPS response in blood and brain was blunted in n-3 offspring. Cerebral angioarchitecture analysis revealed modified vessel complexity after LPS. Thus, n-3-enriched maternal diet partially prevents imbalance in homeostatic processes and alters inflammation rather than affects brain vascularization during early life. Importantly, maternal diet may presage offspring neurovascular outcomes later in life.

Long-term impact of maternal obesity on the gliovascular unit and ephrin signaling in the hippocampus of adult offspring.

BACKGROUND: Children born to obese mothers are at increased risk of developing mood disorders and cognitive impairment. Experimental studies have reported structural changes in the brain such as the gliovascular unit as well as activation of neuroinflammatory cells as a part of neuroinflammation processing in aged offspring of obese mothers. However, the molecular mechanisms linking maternal obesity to poor neurodevelopmental outcomes are not well established. The ephrin system plays a major role in a variety of cellular processes including cell-cell interaction, synaptic plasticity, and long-term potentiation. Therefore, in this study we determined the impact of maternal obesity in pregnancy on cortical, hippocampal development, vasculature and ephrin-A3/EphA4-signaling, in the adult offspring in mice. METHODS: Maternal obesity was induced in mice by a high fat/high sugar Western type of diet (HF/HS). We collected brain tissue (prefrontal cortex and hippocampus) from 6-month-old offspring of obese and lean (control) dams. Hippocampal volume, cortical thickness, myelination of white matter, density of astrocytes and microglia in relation to their activity were analyzed using 3-D stereological quantification. mRNA expression of ephrin-A3, EphA4 and synaptic markers were measured by qPCR in the brain tissue. Moreover, expression of gap junction protein connexin-43, lipocalin-2, and vascular CD31/Aquaporin 4 were determined in the hippocampus by immunohistochemistry. RESULTS: Volume of hippocampus and cortical thickness were significantly smaller, and myelination impaired, while mRNA levels of hippocampal EphA4 and post-synaptic density (PSD) 95 were significantly lower in the hippocampus in the offspring of obese dams as compared to offspring of controls. Further analysis of the hippocampal gliovascular unit indicated higher coverage of capillaries by astrocytic end-feet, expression of connexin-43 and lipocalin-2 in endothelial cells in the offspring of obese dams. In addition, offspring of obese dams demonstrated activation of microglia together with higher density of cells, while astrocyte cell density was lower. CONCLUSION: Maternal obesity affects brain size, impairs myelination, disrupts the hippocampal gliovascular unit and decreases the mRNA expression of EphA4 and PSD-95 in the hippocampus of adult offspring. These results indicate that the vasculature-glia cross-talk may be an important mediator of altered synaptic plasticity, which could be a link between maternal obesity and neurodevelopmental/neuropsychiatric disorders in the offspring.

Therapeutic Effect of Nicotinamide Mononucleotide for Hypoxic-Ischemic Brain Injury in Neonatal Mice.

SUMMARY STATEMENT: Neonatal hypoxia-ischemia reduces nicotinamide adenine dinucleotide (NAD+) and SIRT6 levels in the injured hippocampus.Hippocampal high mobility group box-1 (HMGB1) release is significantly increased after neonatal hypoxia-ischemia.Nicotinamide mononucleotide (NMN) treatment normalizes hippocampal NAD+ and SIRT6 levels, with significant decrease in caspase-3 activity and HMGB1 release.NMN improves early developmental behavior, as well as motor and memory function.

Two different isoforms of osteopontin modulate myelination and axonal integrity.

Abnormal myelination underlies the pathology of white matter diseases such as preterm white matter injury and multiple sclerosis. Osteopontin (OPN) has been suggested to play a role in myelination. Murine OPN mRNA is translated into a secreted isoform (sOPN) or an intracellular isoform (iOPN). Whether there is an isoform-specific involvement of OPN in myelination is unknown. Here we generated mouse models that either lacked both OPN isoforms in all cells (OPN-KO) or lacked sOPN systemically but expressed iOPN specifically in oligodendrocytes (OLs-iOPN-KI). Transcriptome analysis of isolated oligodendrocytes from the neonatal brain showed that genes and pathways related to increase of myelination and altered cell cycle control were enriched in the absence of the two OPN isoforms in OPN-KO mice compared to control mice. Accordingly, adult OPN-KO mice showed an increased axonal myelination, as revealed by transmission electron microscopy imaging, and increased expression of myelin-related proteins. In contrast, neonatal oligodendrocytes from OLs-iOPN-KI mice compared to control mice showed differential regulation of genes and pathways related to the increase of cell adhesion, motility, and vasculature development, and the decrease of axonal/neuronal development. OLs-iOPN-KI mice showed abnormal myelin formation in the early phase of myelination in young mice and signs of axonal degeneration in adulthood. These results suggest an OPN isoform-specific involvement, and a possible interplay between the isoforms, in myelination, and axonal integrity. Thus, the two isoforms of OPN need to be separately considered in therapeutic strategies targeting OPN in white matter injury and diseases.

Proteomics identifies lipocalin-2 in neonatal inflammation associated with cerebrovascular alteration in mice and preterm infants.

Staphylococcus (S.) epidermidis is the most common nosocomial coagulase-negative staphylococci infection in preterm infants. Clinical signs of infection are often unspecific and novel markers to complement diagnosis are needed. We investigated proteomic alterations in mouse brain after S. epidermidis infection and in preterm infant blood. We identified lipocalin-2 (LCN2) as a crucial protein associated with cerebrovascular changes and astrocyte reactivity in mice. We further proved that LCN2 protein expression was associated with endothelial cells but not astrocyte reactivity. By combining network analysis and differential expression approaches, we identified LCN2 linked to blood C-reactive protein levels in preterm infants born <28 weeks of gestation. Blood LCN2 levels were associated with similar alterations of cytokines and chemokines in both infected mice and human preterm infants with increased levels of C-reactive protein. This experimental and clinical study suggests that LCN2 may be a marker of preterm infection/inflammation associated with cerebrovascular changes and neuroinflammation.

Neurological Diseases: A Molecular Genetic Perspective.

Neurological disorders (which include a broad spectrum of central nervous system diseases from children to old people) remain among the most compelling illnesses known to humankind [...].

Transcriptome network analysis links perinatal Staphylococcus epidermidis infection to microglia reprogramming in the immature hippocampus.

Staphylococcus epidermidis (S. epidermidis) is the most common nosocomial pathogen in preterm infants and associated with increased risk of cognitive delay, however, underlying mechanisms are unknown. We employed morphological, transcriptomic and physiological methods to extensively characterize microglia in the immature hippocampus following S. epidermidis infection. 3D morphological analysis revealed activation of microglia after S. epidermidis. Differential expression combined with network analysis identified NOD-receptor signaling and trans-endothelial leukocyte trafficking as major mechanisms in microglia. In support, active caspase-1 was increased in the hippocampus and using the LysM-eGFP knock-in transgenic mouse, we demonstrate infiltration of leukocytes to the brain together with disruption of the blood-brain barrier. Our findings identify activation of microglia inflammasome as a major mechanism underlying neuroinflammation following infection. The results demonstrate that neonatal S. epidermidis infection share analogies with S. aureus and neurological diseases, suggesting a previously unrecognized important role in neurodevelopmental disorders in preterm born children.

Effect of multifactorial therapeutic approach on axonal regeneration and cell viability in an in-vitro model of spinal-derived neural injury.

The highly debilitated nature of spinal cord injuries (SCI) creates an inhibitory repair environment that limits the recovery rate and therefore single interventional treatment has been resulted in incomplete recovery. A multifactorial approach that combines several therapeutic approaches may address diverse aspects of SCI pathology and enhance the recovery rate over single therapy. Accordingly, in this study, we aimed to investigate the effect of combined olfactory ensheathing cells (OECs) (to transport trophic factor, mediate immunomodulation, provide a suitable environment for cell survival), G-CSF (to establish a favorable environment for cell survival) and lipopolysaccharide (LPS) (to boost the protective activity of OEC) therapy on the cell viability after a scratch injury caused by a cataract knife on cells in an in-vitro model of spinal-derived neural injury. In this study, we used mixed neuronal-glial cultures, which are widely used for an in vitro study of neuronal damage. Scratch insult was made on cells using a cataract knife. The cells were divided into 8 groups (two control groups with and without olfactory ensheathing cells (OECs) treatment, injury group, three injury groups with single therapy by using super low dose of LPS (SLD-LPS) (100 pg/ml), OEC group, and G-CSF (100 ng/ml) group, and two injury groups with combined therapy (OEC with SLD-LPS and with all three treatments)). We found a significant decrease in the survival rate of injured cells (p < 0.001) 24 h after scratching insult. Our results indicated morphological alterations in cells in the acute phase (1, 2 and 6 h) after injury, with significant increased gap size at 6 h after induction of injury. Our combined therapy, significantly prevented cell death and decreased the size of the gap over time. We found that combined therapy promoted cell survival following spinal injury by providing a neuroprotective environment for cells. Therefore, our findings provide new insight into the combined therapy, which can be considered for promising preclinical therapeutic strategy for SCI toward clinical trials.

Excessive audio-visual stimulation leads to impaired social behaviour with an effect on amygdala: Early life excessive exposure to digital devices in male rats.

Today, the effect of extreme early-life exposure to digital devices is suggested as a risk factor for neurodevelopmental disorders. However, the multitude of factors that influence brain development with subsequent behavioural abnormalities have not been fully elucidated. Herein, we simulated extreme early-life exposure to digital devices in rats by audio and visual stimulation and investigated its effects on autism-related behaviours and brain structural alteration. Male rat pups were exposed to excessive audio-visual stimulation (EAVS) from PND (post-natal day) 12 to PND 35, with and without maternal separation (MS). Autism-related behaviours including abnormal sociability, stereotype behaviours, anxiety and locomotor dysfunction were tested at PND 42. Brain structural alternation was examined by considering the amygdala, mPFC (medial prefrontal cortex) and hippocampal regions while performing 3D quantitative stereological analysis. We found that EAVS led to social behaviour deficit and higher locomotion in rats, which were associated with increases in the number of neurons and volume of the amygdala. We also showed that MS did not exaggerate the effect of extreme sensory stimulation on behaviour and the structure of the brain. This study proposed EAVS in rats as an animal model of early exposure to digital devices for investigating possible neurobiological alternations underlying autistic-like behaviours with an emphasis on the amygdala area.

The Alpha 7 Nicotinic Acetylcholine Receptor Does Not Affect Neonatal Brain Injury.

Inflammation plays a central role in the development of neonatal brain injury. The alpha 7 nicotinic acetylcholine receptor (α7nAChR) can modulate inflammation and has shown promising results as a treatment target in rodent models of adult brain injury. However, little is known about the role of the α7nAChR in neonatal brain injury. Hypoxic-ischemic (HI) brain injury was induced in male and female C57BL/6 mice, α7nAChR knock-out (KO) mice and their littermate controls on postnatal day (PND) 9-10. C57BL/6 pups received i.p. injections of α7nAChR agonist PHA 568487 (8 mg/kg) or saline once daily, with the first dose given directly after HI. Caspase-3 activity and cytokine mRNA expression in the brain was analyzed 24 h after HI. Motor function was assessed 24 and 48 h after HI, and immunohistochemistry was used to assess tissue loss at 24 h and 7 days after HI and microglial activation 7 days after HI. Activation of α7nAChR with the agonist PHA 568487 significantly decreased CCL2/MCP-1, CCL5/RANTES and IL-6 gene expression in the injured brain hemisphere 24 h after HI compared with saline controls in male, but not female, pups. However, α7nAChR activation did not alter caspase-3 activity and TNFα, IL-1ß and CD68 mRNA expression. Furthermore, agonist treatment did not affect motor function (24 or 48 h), neuronal tissue loss (24 h or 7 days) or microglia activation (7 days) after HI in either sex. Knock-out of α7nAChR did not influence neuronal tissue loss 7 days after HI. In conclusion, targeting the α7nAChR in neonatal brain injury shows some effect on dampening acute inflammatory responses in male pups. However, this does not lead to an effect on overall injury outcome.

Geometrical modelling of neuronal clustering and development.

The dynamic geometry of neuronal development is an essential concept in theoretical neuroscience. We aimed to design a mathematical model which outlines stepwise in an innovative form and designed to model neuronal development geometrically and modelling spatially the neuronal-electrical field interaction. We demonstrated flexibility in forming the cell and its nucleus to show neuronal growth from inside to outside that uses a fractal cylinder to generate neurons (pyramidal/sphere) in form of mathematically called 'surface of revolution'. Furthermore, we verified the effect of the adjacent neurons on a free branch from one-side, by modelling a 'normal vector surface' that represented a group of neurons. Our model also indicated how the geometrical shapes and clustering of the neurons can be transformed mathematically in the form of vector field that is equivalent to the neuronal electromagnetic activity/electric flux. We further simulated neuronal-electrical field interaction that was implemented spatially using Van der Pol oscillator and taking Laplacian vector field as it reflects biophysical mechanism of neuronal activity and geometrical change. In brief, our study would be considered a proper platform and inspiring modelling for next more complicated geometrical and electrical constructions.

Sex-Dependent Gliovascular Interface Abnormality in the Hippocampus following Postnatal Immune Activation in Mice.

The neuro-gliovascular unit is a crucial structure for providing a balanced well-functioning environment for neurons and their synapses. Activation of the immune system during the developmental period is believed to affect the gliovascular unit, which may trigger neurodevelopmental and neurological/neuropsychiatric diseases. In this study, we hypothesized that vulnerability of the male brain to a neonatal insult was conditioned by sex-dependent differences in the impairment of the hippocampal gliovascular unit. Male and female C57BL/6J pups received lipopolysaccharide (LPS) (1 mg/kg) or saline on postnatal day (P) 5. Brains were collected at P12 and morphological quantifications of hippocampal fibrillary glial acid protein (GFAP+) astrocytes and ionized calcium-binding adaptor molecule 1 protein (Iba1+) microglia were performed by using 3-D image analysis together with measuring the length of CD31+ and aquaporin-4 (AQP4+) vessels. We found a significant increase in the length of CD31+ capillaries in the male LPS group compared to the saline group; however, coverage of capillaries by astrocytic end-feet (AQP4+) was significantly reduced. In contrast, there was a significant increase in AQP4+ capillary length in female pups 1 week after LPS injection. GFAP+ astrocytes via morphological changes in the hippocampus showed significant enhancement in the activity 1 week following LPS injection in male mice. We propose that neonatal inflammation could induce susceptibility to neurodevelopmental disorders through modification of hippocampal gliovascular interface in a sex-dependent manner.

An Optimized and Detailed Step-by-Step Protocol for the Analysis of Neuronal Morphology in Golgi-Stained Fetal Sheep Brain.

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure gray matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi-staining protocols have been optimized, and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi-stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semiautomated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking, and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) of gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (µm), branch number, branch order, and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density, and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labor-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

Reelin cells and sex-dependent synaptopathology in autism following postnatal immune activation.

BACKGROUND AND PURPOSE: Autism spectrum disorders (ASD) are heterogeneous neurodevelopmental disorders with considerably increased risk in male infants born preterm and with neonatal infection. Here, we investigated the role of postnatal immune activation on hippocampal synaptopathology by targeting Reelin+ cells in mice with ASD-like behaviours. EXPERIMENTAL APPROACH: C57/Bl6 mouse pups of both sexes received lipopolysaccharide (LPS, 1 mg·kg-1 ) on postnatal day (P) 5. At P45, animal behaviour was examined by marble burying and sociability test, followed by ex vivo brain MRI diffusion kurtosis imaging (DKI). Hippocampal synaptogenesis, number and morphology of Reelin+ cells, and mRNA expression of trans-synaptic genes, including neurexin-3, neuroligin-1, and cell-adhesion molecule nectin-1, were analysed at P12 and P45. KEY RESULTS: Social withdrawal and increased stereotypic activities in males were related to increased mean diffusivity on MRI-DKI and overgrowth in hippocampus together with retention of long-thin immature synapses on apical dendrites, decreased volume and number of Reelin+ cells as well as reduced expression of trans-synaptic and cell-adhesion molecules. CONCLUSION AND IMPLICATIONS: The study provides new insights into sex-dependent mechanisms that may underlie ASD-like behaviour in males following postnatal immune activation. We identify GABAergic interneurons as core components of dysmaturation of excitatory synapses in the hippocampus following postnatal infection and provide cellular and molecular substrates for the MRI findings with translational value.