Single-cell atlas reveals meningeal leukocyte heterogeneity in the developing mouse brain.

The meninges are important for brain development and pathology. Using single-cell RNA sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared with their adult counterparts. We also identified novel meningeal microglia-like cell populations that may participate in white matter development. Early after the hypoxic-ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell resolution view of the importance of meningeal leukocytes at the early stage of development in health and disease.

Immune-Neurovascular Interactions in Experimental Perinatal and Childhood Arterial Ischemic Stroke.

Emerging clinical and preclinical data have demonstrated that the pathophysiology of arterial ischemic stroke in the adult, neonates, and children share similar mechanisms that regulate brain damage but also have distinct molecular signatures and involved cellular pathways due to the maturational stage of the central nervous system and the immune system at the time of the insult. In this review, we discuss similarities and differences identified thus far in rodent models of 2 different diseases-neonatal (perinatal) and childhood arterial ischemic stroke. In particular, we review acquired knowledge of the role of resident and peripheral immune populations in modulating outcomes in models of perinatal and childhood arterial ischemic stroke and the most recent and relevant findings in relation to the immune-neurovascular crosstalk, and how the influence of inflammatory mediators is dependent on specific brain maturation stages. Finally, we discuss the current state of treatments geared toward age-appropriate therapies that signal via the immune-neurovascular interaction and consider sex differences to achieve successful translation.

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.

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.

Treatment with RNase alleviates brain injury but not neuroinflammation in neonatal hypoxia/ischemia.

There is a need for new treatments to reduce brain injuries derived from neonatal hypoxia/ischemia. The only viable option used in the clinic today in infants born at term is therapeutic hypothermia, which has a limited efficacy. Treatments with exogenous RNase have shown great promise in a range of different adult animal models including stroke, ischemia/reperfusion injury, or experimental heart transplantation, often by conferring vascular protective and anti-inflammatory effects. However, any neuroprotective function of RNase treatment in the neonate remains unknown. Using a well-established model of neonatal hypoxic/ischemic brain injury, we evaluated the influence of RNase treatment on RNase activity, gray and white matter tissue loss, blood-brain barrier function, as well as levels and expression of inflammatory cytokines in the brain up to 6 h after the injury using multiplex immunoassay and RT-PCR. Intraperitoneal treatment with RNase increased RNase activity in both plasma and cerebropinal fluids. The RNase treatment resulted in a reduction of brain tissue loss but did not affect the blood-brain barrier function and had only a minor modulatory effect on the inflammatory response. It is concluded that RNase treatment may be promising as a neuroprotective regimen, whereas the mechanistic effects of this treatment appear to be different in the neonate compared to the adult and need further investigation.

Novel biomarkers of preterm brain injury from blood transcriptome in sheep model of intrauterine asphyxia.

BACKGROUND: Infants born preterm have a higher incidence of neurological deficits. A key step in finding effective treatments is to identify biomarkers that reliably predict outcome. METHODS: Following umbilical cord occlusion (UCO) in pregnant sheep, whole fetal blood RNA was sequenced pre- and post-UCO, brain injury outcome was determined by battery of neuropathology scoring and the transcriptome signature correlated to the degree of brain injury. Additionally, we developed a novel analytical procedure to deduce cell blood composition over time. RESULTS: Sixty-one genes were identified with significant altered expression after UCO. In pre-UCO blood, the level of three mRNAs (Trex2, Znf280b, novel miRNA) and in post-UCO, four mRNAs (Fam184a, Angptl2, novel lincRNA and an unknown protein-coding gene) were associated to brain injury (FDR < 0.01). Several of these mRNAs are related to inflammation and angiogenesis. Pathway analysis highlighted genes playing a role in perinatal death and growth failure. Results also indicate that several leukocyte populations undergo significant changes after UCO. CONCLUSION: We have used a whole transcriptomic approach to uncover novel biomarkers in fetal blood that correlate to neuropathology in the preterm sheep brain. The current data forms a basis for future studies to investigate mechanisms of these mRNAs in the injury progression. IMPACT: Trend analysis of genes following asphyxia reveal a group of genes associated with perinatal death and growth failure. Several pre-asphyxia transcripts were associated to brain injury severity suggesting genomic susceptibility to injury. Several post-asphyxia transcripts were correlated to brain injury severity, thus, serve as potential novel biomarkers of injury outcome. Successfully adaptation of cell profiling algorithms suggests significant changes in blood cell composition following asphyxia.

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.

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.

Scavenger receptor CD36 governs recruitment of myeloid cells to the blood-CSF barrier after stroke in neonatal mice.

BACKGROUND: Ischemic stroke induces the activation and recruitment of peripheral leukocytes to the injured brain. These cells can infiltrate the brain through multiple routes, either by penetrating blood-brain barrier or via blood-CSF barriers at the meninges or the choroid plexus (CP). We previously showed that myeloid cell trafficking via the CP occurs early after neonatal arterial stroke and modulates injury. CD36 is a receptor that mediates function of endothelial cells and cells of the monocyte lineage under various neurodegenerative conditions and can influence brain injury after neonatal stroke. Here we asked whether CD36 impacts injury by altering leukocyte trafficking through the CP in neonatal mice subjected to transient middle cerebral artery occlusion (tMCAO). METHODS: In neonatal mice with intact or globally disrupted CD36 signalling (CD36 KO), we characterized the phenotypes of myeloid cells by flow cytometry and the underlying gene expression signatures in the CPs contralateral and ipsilateral to tMCAO by RNA sequencing analyses, focussing on early post-reperfusion time window. RESULTS: Flow cytometry in the isolated CPs revealed that CD36 mediates stepwise recruitment of myeloid cells to the CP ipsilateral to tMCAO early after reperfusion, with a predominant increase first in inflammatory monocyte subsets and neutrophils followed by patrolling monocytes. RNA sequencing analyses demonstrated marked changes in gene expression in the CP ipsilateral compared to the CP contralateral to tMCAO in wild type mice. Changes were further modified by lack of CD36, including distinction in several clusters of genes involved in inflammatory, metabolic and extracellular matrix signalling in the CP ipsilateral to tMCAO. CONCLUSION: Altogether, our data suggest cooperation between blood-CSF-brain interface via the CP through CD36-mediated signalling following neonatal stroke with a key role for inflammatory monocytes and neutrophils.

Administration of cyclic glycine-proline during infancy improves adult spatial memory, astrocyte plasticity, vascularization and GluR-1 expression in rats.

Cyclic glycine-proline (cGP) is a natural nutrient of breast milk and plays a role in regulating the function of insulin-like growth factor-1 (IGF-1). IGF-1 function is essential for post-natal brain development and adult cognitive function. We evaluated the effects of cGP on spatial memory and histological changes in the hippocampus of the adult rats following infancy administration. Infant rats were treated with either cGP or saline between post-natal days 8 and 22 via oral administration to lactating dams. The spatial memory was evaluated between post-natal days 70 and 75 using Morris water maze tests. The changes of capillaries, astrocytes, synaptophysin and glutamate receptor-1 were examined in the CA1 stratum radiatum of the hippocampus. Compared to saline-treated group, cGP-treated group showed higher path efficiency of entry and lower average heading errors to the platform zone. cGP-treated group also showed longer, larger and more astrocytic processes, more capillaries and higher glutamate receptor-1 expression. The rats made less average heading error to the platform zone have more capillaries, larger and longer astrocytic branches. Thus cGP treatment/supplementation during infancy moderately improved adulthood spatial memory. This long-lasting effect of cGP on memory could be mediated via promoting astrocytic plasticity, vascularization and glutamate trafficking. Therefore, cGP may have a role in regulating IGF-1 function during brain development.

Neuroprotection offered by mesenchymal stem cells in perinatal brain injury: Role of mitochondria, inflammation, and reactive oxygen species.

Preclinical studies have shown that mesenchymal stem cells have a positive effect in perinatal brain injury models. The mechanisms that cause these neurotherapeutic effects are not entirely intelligible. Mitochondrial damage, inflammation, and reactive oxygen species are considered to be critically involved in the development of injury. Mesenchymal stem cells have immunomodulatory action and exert mitoprotective effects which attenuate production of reactive oxygen species and promote restoration of tissue function and metabolism after perinatal insults. This review summarizes the present state, the underlying causes, challenges and possibilities for effective clinical translation of mesenchymal stem cell therapy.

Galectin-3 Modulates Microglia Inflammation in vitro but Not Neonatal Brain Injury in vivo under Inflammatory Conditions.

Microglia may contribute to injury but may also have neuroprotective properties. Galectin-3 has immunomodulatory properties that may affect the microglia phenotype and subsequent development of injury. Galectin-3 contributes to experimental hypoxic-ischemic (HI) injury in the neonatal brain, but it is unclear if galectin-3 has similar effects on infectious and sterile inflammation. Thus, we investigated the effect of galectin-3 on microglia in vitro under normal as well as infectious and sterile inflammatory conditions, and the effect of galectin-3 on neonatal brain injury following an infectious challenge in vivo. Conditions mimicking infectious or sterile inflammation were evaluated in primary microglia cell cultures from newborn mice, using LPS (10 ng/mL) and TNF-α (100 ng/mL). The response to galectin-3 was tested alone or together with LPS or TNF-α. Supernatants were collected 24 h after treatment and analyzed for 23 inflammatory mediators including pro- and anti-inflammatory cytokines and chemokines using multiplex protein analysis, as well as ELISA for MCP-1 and insulin-like growth factor (IGF)-1. Phosphorylation of proteins (AKT, ERK1/2, IκB-α, JNK, and p38) was determined in microglia cells. Neonatal brain injury was induced by a combination of LPS and HI (LPS + HI) in postnatal day 9 transgenic mice lacking functional galectin-3 and wild-type controls. LPS and TNF-α induced pro-inflammatory (9/11 vs. 9/10) and anti-inflammatory (6/6 vs. 2/6) cytokines, as well as chemokines (6/6 vs. 4/6) in a similar manner, except generally lower amplitude of the TNF-α-induced response. Galectin-3 alone had no effect on any of the proteins analyzed. Galectin-3 reduced the LPS- and TNF-α-induced microglia response for cytokines, chemokines, and phosphorylation of IκB-α. LPS decreased baseline IGF-1 levels, and the levels were restored by galectin-3. Brain injury or microglia response after LPS + HI was not affected by galectin-3 deficiency. Galectin-3 has no independent effect on microglia but modulates inflammatory activation in vitro. The effect was similar under infectious and sterile inflammatory conditions, suggesting that galectin-3 regulates inflammation not just by binding to LPS or toll-like receptor-4. Galectin-3 restores IGF-1 levels reduced by LPS-induced inflammation, suggesting a potential protective effect on infectious injury. However, galectin-3 deficiency did not affect microglia activation and was not beneficial in an injury model encompassing an infectious challenge.

Growth-differentiation-factor 15 levels in obese and healthy pregnancies: Relation to insulin resistance and insulin secretory function.

OBJECTIVE/AIM: Growth-differentiation-factor 15 (GDF15) has been suggested to improve or protect beta cell function. During pregnancy, beta cell numbers and function increase to overcome the natural rise in insulin resistance during gestation. In this study, we longitudinally measured serum GDF15 levels during and after pregnancy in women of normal weight (NW) and in women with obesity (OB) and explored associations between GDF15 and changes in beta cell function by homeostatic model assessment (HOMA). METHODS: The cohort participants were 38 NW (BMI 22.3 ± 1.7) and 35 OB (BMI 35.8 ± 4.2). Blood was sampled and body composition measured at each trimester (T1, T2, and T3) and at 6, 12 and 18 months postpartum. Fasting glucose, insulin and GDF15 were measured, and HOMA for insulin resistance (HOMA-IR) and beta cell function (HOMA-B) determined. RESULTS: GDF15 levels increased significantly each trimester and were ~200-fold higher at T3 than in the nonpregnant postpartum state. GDF15 was higher in NW than OB during pregnancy, but was reversed after pregnancy with a significant interaction effect. GDF15 correlated inversely with BMI and fat-free mass at T3. Low GDF15 was associated with lower incidence of nausea and with carrying a male foetus. The pregnancy induced increase in GDF15 associated with increased HOMA-B in OB and with reduced fasting glucose in all women. CONCLUSION: Large gestational upregulation of GDF15 levels may help increase insulin secretory function to overcome pregnancy-induced insulin resistance.

Association between inflammatory response and outcome after subarachnoid haemorrhage.

OBJECTIVES: Recent reports suggest an association between the inflammatory response after aneurysmal subarachnoid haemorrhage (aSAH) and patients' outcome. The primary aim of this study was to identify a potential association between the inflammatory response after aSAH and 1-year outcome. The secondary aim was to investigate whether the inflammatory response after aSAH could predict the development of delayed cerebral ischaemia (DCI). MATERIALS AND METHODS: This prospective observational pilot study included patients with an aSAH admitted to Sahlgrenska University Hospital, Gothenburg, Sweden, between May 2015 and October 2016. The patients were stratified according to the extended Glasgow Outcome Scale (GOSE) as having an unfavourable (score: 1-4) or favourable outcome (score: 5-8). Furthermore, patients were stratified depending on development of DCI or not. Patient data and blood samples were collected and analysed at admission and after 10 days. RESULTS: Elevated serum concentrations of inflammatory markers such as tumour necrosis factor-α and interleukin (IL)-6, IL-1Ra, C-reactive protein and intercellular adhesion molecule-1 were detected in patients with unfavourable outcome. When adjustments for Glasgow coma scale were made, only IL-1Ra remained significantly associated with poor outcome (p = 0.012). The inflammatory response after aSAH was not predictive of the development of DCI. CONCLUSION: Elevated serum concentrations of inflammatory markers were associated with poor neurological outcome 1-year after aSAH. However, inflammatory markers are affected by many clinical events, and when adjustments were made, only IL-1Ra remained significantly associated with poor outcome. The robustness of these results needs to be tested in a larger trial.

Dual Profile of Environmental Enrichment and Autistic-Like Behaviors in the Maternal Separated Model in Rats.

BACKGROUND: Environmental Enrichment (EE) has been suggested as a possible therapeutic intervention for neurodevelopmental disorders such as autism. Although the benefits of this therapeutic method have been reported in some animal models and human studies, the unknown pathophysiology of autism as well as number of conflicting results, urge for further examination of the therapeutic potential of EE in autism. Therefore, the aim of this study was to examine the effects of environmental enrichment on autism-related behaviors which were induced in the maternal separation (MS) animal model. MATERIAL AND METHODS: Maternally separated (post-natal day (PND) 1-14, 3h/day) and control male rats were at weaning (PND21) age equally divided into rats housed in enriched environment and normal environment. At adolescence (PND42-50), the four groups were behaviorally tested for direct social interaction, sociability, repetitive behaviors, anxiety behavior, and locomotion. Following completion of the behavioral tests, the blood and brain tissue samples were harvested in order to assess plasma level of brain derived neurotrophic factor (BDNF) and structural plasticity of brain using ELISA and stereological methods respectively. RESULTS: We found that environmental enrichment reduced repetitive behaviors but failed to improve the impaired sociability and anxiety behaviors which were induced by maternal separation. Indeed, EE exacerbated anxiety and social behaviors deficits in association with increased plasma BDNF level, larger volume of the hippocampus and infra-limbic region and higher number of neurons in the infra-limbic area (p < 0.05). Conclusion: We conclude that environmental enrichment has a significant improvement effect on the repetitive behavior as one of the core autistic-like behaviors induced by maternal separation but has negative effect on the anxiety and social behaviors which might have been modulated by BDNF.

Vancomycin Is Protective in a Neonatal Mouse Model of Staphylococcus epidermidis-Potentiated Hypoxic-Ischemic Brain Injury.

Infection is correlated with increased risk of neurodevelopmental sequelae in preterm infants. In modeling neonatal brain injury, Toll-like receptor agonists have often been used to mimic infections and induce inflammation. Using the most common cause of bacteremia in preterm infants, Staphylococcus epidermidis, we present a more clinically relevant neonatal mouse model that addresses the combined effects of bacterial infection together with subsequent hypoxic-ischemic brain insult. Currently, there is no neuroprotective treatment for the preterm population. Hence, we tested the neuroprotective effects of vancomycin with and without adjunct therapy using the anti-inflammatory agent pentoxifylline. We characterized the effects of S. epidermidis infection on the inflammatory response in the periphery and the brain, as well as the physiological changes in the central nervous system that might affect neurodevelopmental outcomes. Intraperitoneal injection of postnatal day 4 mice with a live clinical isolate of S. epidermidis led to bacteremia and induction of proinflammatory cytokines in the blood, as well as transient elevations of neutrophil and monocyte chemotactic cytokines and caspase 3 activity in the brain. When hypoxia-ischemia was induced postinfection, more severe brain damage was observed in infected animals than in saline-injected controls. This infection-induced inflammation and potentiated brain injury was inoculum dose dependent and was alleviated by the antibiotic vancomycin. Pentoxifylline did not provide any additional neuroprotective effect. Thus, we show for the first time that live S. epidermidis potentiates hypoxic-ischemic preterm brain injury and that peripheral inhibition of inflammation with antibiotics, such as vancomycin, reduces the extent of brain injury.

Longitudinal changes in adipokines and free leptin index during and after pregnancy in women with obesity.

OBJECTIVE: Detailed data on adipokines and body composition during and after pregnancy in women of different BMI categories are lacking. Furthermore, adipokine regulation during pregnancy and the factors contributing to gestational insulin resistance are not completely understood. The objective was to longitudinally determine adipokine levels, body composition, and insulin sensitivity during and after pregnancy in women of healthy weight (HW) and with obesity (OB), and identify factors associated with insulin resistance. DESIGN: Women (30 HW, 19 OB) underwent blood sampling and body composition examination, by air-displacement plethysmography, longitudinally during pregnancy (trimesters 1, 2, 3) and after pregnancy (6, 12, 18 months postpartum). Serum leptin, soluble leptin receptor (sOB-R), and adiponectin levels were measured and free leptin index (FLI) and homeostatic model assessment of insulin resistance (HOMA-IR) determined. RESULTS: Fat mass and leptin increased during pregnancy in the HW (p < 0.01) but not in the OB group. sOB-R increased during pregnancy in both groups (p < 0.001). Thus, FLI was unchanged in HW throughout pregnancy but reduced in OB (p = 0.001), although consistently higher in OB. Adiponectin decreased in both groups during pregnancy (p < 0.001 for HW, p = 0.01 for OB). After pregnancy, adiponectin increased in both groups, but more markedly in OB where it reached trimester 1 levels. Multivariable regression identified FLI as the variable most strongly associated with HOMA-IR in all trimesters, but not after pregnancy. CONCLUSIONS: Leptin, sOB-R, adiponectin, and FLI undergo marked changes during and after pregnancy with differences in women of different BMI. We suggest that leptin activity is regulated by its soluble receptor and that this is an important factor for optimizing fat mass and insulin sensitivity during pregnancy.

Microbial invasion of the amniotic cavity is associated with impaired cognitive and motor function at school age in preterm children.

BACKGROUND: Chorioamnionitis is an important cause of preterm delivery. Data on neurodevelopmental outcome in exposed infants are inconsistent due to difficulties in diagnosing intrauterine infection/inflammation and lack of detailed long-term follow-up. We investigate cognitive and motor function in preterm infants at early school age and relate the findings to bacteria in amniotic fluid obtained by amniocentesis (microbial invasion of the amniotic cavity (MIAC)) or placenta findings of histological chorioamnionitis (HCA) or fetal inflammatory response syndrome (FIRS). METHOD: Sixty-six infants with gestational age <34 weeks at birth and without major disabilities were assessed using WISC-III and the Bruininks-Oseretsky Test of Motor Proficiency. Results were corrected for gestational age and sex. RESULTS: Children exposed to MIAC had significantly lower scores for full-scale IQ and verbal IQ compared to the non-MIAC group and the difference in full-scale IQ remained after correction for confounding factors. The MIAC group had also significantly lower motor scores after correction. In contrast, motor function was not affected in infants exposed to HCA or FIRS and differences between groups for cognitive scores were lost after corrections. CONCLUSION: Exposure to bacteria in amniotic fluid is associated with lower motor and cognitive scores in school age preterm infants without major disabilities.

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo.

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro-inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)-expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS-mediated Toll-like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi-1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi-1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi-1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi-1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro-inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation.