Impact of Fetal Growth Restriction on the Neonatal Microglial Proteome in the Rat.

Microglial activation is a key modulator of brain vulnerability in response to intra-uterine growth restriction (IUGR). However, the consequences of IUGR on microglial development and the microglial proteome are still unknown. We used a model of IUGR induced by a gestational low-protein diet (LPD) in rats. Microglia, isolated from control and growth-restricted animals at P1 and P4, showed significant changes in the proteome between the two groups. The expression of protein sets associated with fetal growth, inflammation, and the immune response were significantly enriched in LPD microglia at P1 and P4. Interestingly, upregulation of protein sets associated with the oxidative stress response and reactive oxygen species production was observed at P4 but not P1. During development, inflammation-associated proteins were upregulated between P1 and P4 in both control and LPD microglia. By contrast, proteins associated with DNA repair and senescence pathways were upregulated in only LPD microglia. Similarly, protein sets involved in protein retrograde transport were significantly downregulated in only LPD microglia. Overall, these data demonstrate significant and multiple effects of LPD-induced IUGR on the developmental program of microglial cells, leading to an abnormal proteome within the first postnatal days.

Genetic Deletion of mGlu3 Metabotropic Glutamate Receptors Amplifies Ischemic Brain Damage and Associated Neuroinflammation in Mice.

Backgroud: Type-3 metabotropic glutamate (mGlu3) receptors are found in both neurons and glial cells and regulate synaptic transmission, astrocyte function, and microglial reactivity. Here we show that the genetic deletion of mGlu3 receptors amplifies ischemic brain damage and associated neuroinflammation in adult mice. An increased infarct size was observed in mGlu3-/- mice of both CD1 and C57Black strains 24 h following a permanent occlusion of the middle cerebral artery (MCA) as compared to their respective wild-type (mGlu3+/+ mice) counterparts. Increases in the expression of selected pro-inflammatory genes including those encoding interleukin-1ß, type-2 cycloxygenase, tumor necrosis factor-α, CD86, and interleukin-6 were more prominent in the peri-infarct region of mGlu3-/- mice. In contrast, the expression of two genes associated with the anti-inflammatory phenotype of microglia (those encoding the mannose-1-phosphate receptor and the α-subunit of interleukin-4 receptor) and the gene encoding the neuroprotective factor, glial cell line-derived neurotrophic factor, was enhanced in the peri-infarct region of wild-type mice, but not mGlu3-/- mice, following MCA occlusion. In C57Black mice, the genetic deletion of mGlu3 receptors worsened the defect in the paw placement test as assessed in the contralateral forepaw at short times (4 h) following MCA occlusion. These findings suggest that mGlu3 receptors are protective against ischemic brain damage and support the way to the use of selective mGlu3 receptor agonists or positive allosteric modulators in experimental animal models of ischemic stroke.

Bedside functional monitoring of the dynamic brain connectivity in human neonates.

Clinicians have long been interested in functional brain monitoring, as reversible functional losses often precedes observable irreversible structural insults. By characterizing neonatal functional cerebral networks, resting-state functional connectivity is envisioned to provide early markers of cognitive impairments. Here we present a pioneering bedside deep brain resting-state functional connectivity imaging at 250-µm resolution on human neonates using functional ultrasound. Signal correlations between cerebral regions unveil interhemispheric connectivity in very preterm newborns. Furthermore, fine-grain correlations between homologous pixels are consistent with white/grey matter organization. Finally, dynamic resting-state connectivity reveals a significant occurrence decrease of thalamo-cortical networks for very preterm neonates as compared to control term newborns. The same method also shows abnormal patterns in a congenital seizure disorder case compared with the control group. These results pave the way to infants' brain continuous monitoring and may enable the identification of abnormal brain development at the bedside.

mGlu3 receptor regulates microglial cell reactivity in neonatal rats.

BACKGROUND: Perinatal inflammation is a key factor of brain vulnerability in neonates born preterm or with intra-uterine growth restriction (IUGR), two leading conditions associated with brain injury and responsible for neurocognitive and behavioral disorders. Systemic inflammation is recognized to activate microglia, known to be the critical modulators of brain vulnerability. Although some evidence supports a role for metabotropic glutamate receptor 3 (mGlu3 receptor) in modulation of neuroinflammation, its functions are still unknown in the developing microglia. METHODS: We used a double-hit rat model of perinatal brain injury induced by a gestational low-protein diet combined with interleukin-1ß injections (LPD/IL-1ß), mimicking both IUGR and prematurity-related inflammation. The effect of LPD/IL-1ß on mGlu3 receptor expression and the effect of mGlu3 receptor modulation on microglial reactivity were investigated using a combination of pharmacological, histological, and molecular and genetic approaches. RESULTS: Exposure to LPD/IL-1ß significantly downregulated Grm3 gene expression in the developing microglia. Both transcriptomic analyses and pharmacological modulation of mGlu3 receptor demonstrated its central role in the control of inflammation in resting and activated microglia. Microglia reactivity to inflammatory challenge induced by LPD/IL-1ß exposure was reduced by an mGlu3 receptor agonist. Conversely, both specific pharmacological blockade, siRNA knock-down, and genetic knock-out of mGlu3 receptors mimicked the pro-inflammatory phenotype observed in microglial cells exposed to LPD/IL-1ß. CONCLUSIONS: Overall, these data show that Grm3 plays a central role in the regulation of microglial reactivity in the immature brain. Selective pharmacological activation of mGlu3 receptors may prevent inflammatory-induced perinatal brain injury.

Perinatal Stress Programs Sex Differences in the Behavioral and Molecular Chronobiological Profile of Rats Maintained Under a 12-h Light-Dark Cycle.

Stress and the circadian systems play a major role in an organism's adaptation to environmental changes. The adaptive value of the stress system is reactive while that of the circadian system is predictive. Dysfunctions in these two systems may account for many clinically relevant disorders. Despite the evidence that interindividual differences in stress sensitivity and in the functioning of the circadian system are related, there is limited integrated research on these topics. Moreover, sex differences in these systems are poorly investigated. We used the perinatal stress (PRS) rat model, a well-characterized model of maladaptive programming of reactive and predictive adaptation, to monitor the running wheel behavior in male and female adult PRS rats, under a normal light/dark cycle as well as in response to a chronobiological stressor (6-h phase advance/shift). We then analyzed across different time points the expression of genes involved in circadian clocks, stress response, signaling, and glucose metabolism regulation in the suprachiasmatic nucleus (SCN). In the unstressed control group, we found a sex-specific profile that was either enhanced or inverted by PRS. Also, PRS disrupted circadian wheel-running behavior by inducing a phase advance in the activity of males and hypoactivity in females and increased vulnerability to chronobiological stress in both sexes. We also observed oscillations of several genes in the SCN of the unstressed group in both sexes. PRS affected males to greater extent than females, with PRS males displaying a pattern similar to unstressed females. Altogether, our findings provide evidence for a specific profile of dysmasculinization induced by PRS at the behavioral and molecular level, thus advocating the necessity to include sex as a biological variable to study the set-up of circadian system in animal models.

Pain, Parental Involvement, and Oxytocin in the Neonatal Intensive Care Unit.

Preterm infants (PTI) typically experience many painful and stressful procedures or events during their first weeks of life in a neonatal intensive care unit, and these can profoundly impact subsequent brain development and function. Several protective interventions during this sensitive period stimulate the oxytocin system, reduce pain and stress, and improve brain development. This review provides an overview of the environmental risk factors experienced by PTI during hospitalization, with a focus on the effects of pain, and early maternal separation. We also describe the long-term adverse effects of the simultaneous experiences of pain and maternal separation, and the potential beneficial effects of maternal vocalizations, parental contact, and several related processes, which appear to be mediated by the oxytocin system.

Ultrafast Doppler for neonatal brain imaging.

The emergence of functional neuroimaging has dramatically accelerated our understanding of the human mind. The advent of functional Magnetic Resonance Imaging paved the way for the next decades' major discoveries in neuroscience and today remains the "gold standard" for deep brain imaging. Recent improvements in imaging technology have been somewhat limited to incremental innovations of mature techniques instead of breakthroughs. Recently, the use of ultrasonic plane waves transmitted at ultrafast frame rates was shown to highly increase Doppler ultrasound sensitivity to blood flows in small vessels in rodents. By identifying regions of brain activation through neurovascular coupling, Ultrafast Doppler was entering into the world of preclinical neuroimaging. The combination of many advantages, including high spatio-temporal resolution, deep penetration, high sensitivity and portability provided unique information about brain function. Recently, Ultrafast Doppler imaging was found able to non-invasively image the spatial and temporal dynamics of microvascular changes during seizures and interictal periods with an unprecedented resolution at bedside. This review summarizes the technical basis, the added value and the clinical perspectives provided by this new brain imaging modality that could create a breakthrough in the knowledge of brain hemodynamics, brain insult, and neuroprotection.

Oxytocin receptor agonist reduces perinatal brain damage by targeting microglia.

Prematurity and fetal growth restriction (FGR) are frequent conditions associated with adverse neurocognitive outcomes. We have previously identified early deregulation of genes controlling neuroinflammation as a putative mechanism linking FGR and abnormal trajectory of the developing brain. While the oxytocin system was also found to be impaired following adverse perinatal events, its role in the modulation of neuroinflammation in the developing brain is still unknown. We used a double-hit rat model of perinatal brain injury induced by gestational low protein diet (LPD) and potentiated by postnatal injections of subliminal doses of interleukin-1ß (IL1ß) and a zebrafish model of neuroinflammation. Effects of the treatment with carbetocin, a selective, long lasting, and brain diffusible oxytocin receptor agonist, have been assessed using a combination of histological, molecular, and functional tools in vivo and in vitro. In the double-hit model, white matter inflammation, deficient myelination, and behavioral deficits have been observed and the oxytocin system was impaired. Early postnatal supplementation with carbetocin alleviated microglial activation at both transcriptional and cellular levels and provided long-term neuroprotection. The central anti-inflammatory effects of carbetocin have been shown in vivo in rat pups and in a zebrafish model of early-life neuroinflammation and reproduced in vitro on stimulated sorted primary microglial cell cultures from rats subjected to LPD. Carbetocin treatment was associated with beneficial effects on myelination, long-term intrinsic brain connectivity and behavior. Targeting oxytocin signaling in the developing brain may be an effective approach to prevent neuroinflammation - induced brain damage of perinatal origin.

Modulation of Microglial Activation by Adenosine A2a Receptor in Animal Models of Perinatal Brain Injury.

Neuroinflammation has a key role in the pathogenesis of perinatal brain injury. Caffeine, a nonspecific antagonist of adenosine receptors (ARs), is widely used to treat apnea of prematurity and has been linked to a decrease in the incidence of cerebral palsy in premature infants. The mechanisms explaining its neuroprotective effect have not yet been elucidated. The objective of this study was to characterize the expression of adenosine and ARs in two neonatal rat models of neuroinflammation and to determine the effect of A2aR blockade on microglial activation assessed through inflammatory cytokine gene expression. We have used two rat models of microglial activation: the gestational low protein diet (LPD) model, associated with chronic brain injury, and postnatal ibotenate intracerebral injections, responsible for acute excitotoxicity injury. Adenosine blood levels have been measured by Tandem Mass Spectrometry. The expression of ARs in vivo was assessed using qPCR and immunohistochemistry. In vivo models have been replicated in vitro on primary microglial cell cultures exposed to A2aR agonist CGS-21680 or antagonist SCH-58261. The effects of these treatments have been assessed on the M1/M2 cytokine expressions measured by RT-qPCR. LPD during pregnancy was associated with higher adenosine levels in pups at postnatal day 1 and 4. A2aR mRNA expression was significantly increased in both cortex and magnetically sorted microglial cells from LPD animals compared to controls. CD73 expression, responsible for extracellular production of brain adenosine, was significantly increased in LPD cortex and sorted microglia cells. Moreover, CD73 protein level was increased in ibotenate treated animals. In vitro experiments confirmed that LPD or control microglial cells exposed to ibotenate display an increased expression, at both protein and molecular levels, of A2aR and M1 markers (IL-1ß, IL-6, iNOS, TNFα). This pro-inflammatory profile was significantly reduced by SCH-58261, which reduces M1 markers in both LPD and ibotenate-exposed cells, with no effect on control cells. In the same experimental conditions, a partial increased of M1 cytokines was observed in response to A2aR agonist CGS-21680. These results support the involvement of adenosine and particularly of its receptor A2aR in the regulation of microglia in two different animal models of neuroinflammation.

Modulating the Oxytocin System During the Perinatal Period: A New Strategy for Neuroprotection of the Immature Brain?

Oxytocin is a neurohypophysal hormone known for its activity during labor and its role in lactation. However, the function of oxytocin (OTX) goes far beyond the peripheral regulation of reproduction, and the central effects of OTX have been extensively investigated, since it has been recognized to influence the learning and memory processes. OTX has also prominent effects on social behavior, anxiety, and autism. Interaction between glucocorticoids, OTX, and maternal behavior may have long-term effects on the developmental program of the developing brain subjected to adverse events during pre and perinatal periods. OTX treatment in humans improves many aspects of social cognition and behavior. Its effects on the hypothalamic-pituitary-adrenal axis and inflammation appear to be of interest in neonates because these properties may confer benefits when the perinatal brain has been subjected to injury. Indeed, early life inflammation and abnormal adrenal response to stress have been associated with an abnormal white matter development. Recent investigations demonstrated that OTX is involved in the modulation of microglial reactivity in the developing brain. This review recapitulates state-of-the art data supporting the hypothesis that the OTX system could be considered as an innovative candidate for neuroprotection, especially in the immature brain.

Consequences of a double hit of stress during the perinatal period and midlife in female rats: Mismatch or cumulative effect?

The interplay between experiences during critical developmental periods and later adult life is crucial in shaping individual variability in stress coping strategies. Exposure to stressful events in early life has strongly programs an individual's phenotype and adaptive capabilities. Until now, studies on programming and reversal strategies in early life stress animal models have been essentially limited to males. By using the perinatal stress (PRS) rat model (a model more sensitive to aging changes) in middle-aged females, we investigated the behavioral and endocrine responses following exposure in later life to an unpredictable chronic mild stress (uCMS) condition for six weeks. PRS by itself accelerated the ageing-related-disruption in the estrous cycle and led to reductions in the levels of estradiol. It also reduced motivational and risk-taking behavior in later life, with PRS females being characterized by a reduction in self-grooming in the splash test, in the exploration of the light compartment in the light/dark box test and in the time spent eating a palatable food in the novelty-induced suppression feeding test. PRS females showed impaired regulation of plasma glucose and insulin levels following a glucose challenge, with a hyperglycemic phenotype, and disrupted feedback of the HPA axis after acute stress with respect to controls. Remarkably, all PRS-induced alterations were modified by exposure to the uCMS procedure, thus resulting in a disease-dependent intervention; controls were not affected by uCMS, except for a slight and transient reduction in body weight, while PRS females displayed a reduced body weight gain for the entire duration of the uCMS procedure. Interestingly, the effects of uCMS on PRS females were still observed up to two months after its termination and the females displayed heightened rhythms of locomotor activity and enhanced sensitivity to reward with respect to controls exposed to uCMS. Our findings indicate that many parameters of the PRS female adult phenotype are shaped by both early and later life experiences in a non-additive way. As a consequence, early stressed individuals may be programmed with a more dynamic phenotype than non-stressed individuals.

Reduced maternal behavior caused by gestational stress is predictive of life span changes in risk-taking behavior and gene expression due to altering of the stress/anti-stress balance.

Exposure of the mother to adverse events during pregnancy is known to induce pathological programming of the HPA axis in the progeny, thereby increasing the vulnerability to neurobehavioral disorders. Maternal care plays a crucial role in the programming of the offspring, and oxytocin plays a key role in mother/pup interaction. Therefore, we investigated whether positive modulation of maternal behavior by activation of the oxytocinergic system could reverse the long-term alterations induced by perinatal stress (PRS; gestational restraint stress 3 times/day during the last ten days of gestation) on HPA axis activity, risk-taking behavior in the elevated-plus maze, hippocampal mGlu5 receptor and gene expression in Sprague-Dawley rats. Stressed and control unstressed dams were treated during the first postpartum week with an oxytocin receptor agonist, carbetocin (1 mg/kg, i.p.). Remarkably, reduction of maternal behavior was predictive of behavioral disturbances in PRS rats as well as of the impairment of the oxytocin and its receptor gene expression. Postpartum carbetocin corrected the reduction of maternal behavior induced by gestational stress as well as the impaired oxytocinergic system in the PRS progeny, which was associated with reduced risk-taking behavior. Moreover, postpartum carbetocin had an anti-stress effect on HPA axis activity in the adult PRS progeny and increased hippocampal mGlu5 receptor expression in aging. In conclusion, the activation of the oxytocinergic system in the early life plays a protective role against the programming effect by adverse experiences and could be considered as a novel and powerful potential therapeutic target for stress-related disorders.

Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system.

mGlu5 receptors are involved in mechanisms of activity-dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor-mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor-mediated somatic Ca2+ mobilization, and mGlu3 receptor-mediated long-term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field.

Functional ultrasound imaging of brain activity in human newborns.

Functional neuroimaging modalities are crucial for understanding brain function, but their clinical use is challenging. Recently, the use of ultrasonic plane waves transmitted at ultrafast frame rates was shown to allow for the spatiotemporal identification of brain activation through neurovascular coupling in rodents. Using a customized flexible and noninvasive headmount, we demonstrate in human neonates that real-time functional ultrasound imaging (fUSI) is feasible by combining simultaneous continuous video-electroencephalography (EEG) recording and ultrafast Doppler (UfD) imaging of the brain microvasculature. fUSI detected very small cerebral blood volume variations in the brains of neonates that closely correlated with two different sleep states defined by EEG recordings. fUSI was also used to assess brain activity in two neonates with congenital abnormal cortical development enabling elucidation of the dynamics of neonatal seizures with high spatiotemporal resolution (200 µm for UfD and 1 ms for EEG). fUSI was then applied to track how waves of vascular changes were propagated during interictal periods and to determine the ictal foci of the seizures. Imaging the human brain with fUSI enables high-resolution identification of brain activation through neurovascular coupling and may provide new insights into seizure analysis and the monitoring of brain function.

Relocation stress induces short-term fecal cortisol increase in Tonkean macaques (Macaca tonkeana).

The level of glucocorticoids, especially if obtained from noninvasive sampling, can be used as an index of animal well-being, allowing evaluation of the animal's response to environmental modifications. Despite evidence that these hormones play a relevant role in energy metabolism regulation in perceived or real stress events, little is known regarding the factors that could modify the capability of animals to cope with relocation events. The aim of this research was to assess fecal cortisol metabolite concentrations before, during and after acute stress (transfer and relocation event) in two well-established social groups of Tonkean macaques (Macaca tonkeana). The results showed that the fecal levels of cortisol increased in individuals of both groups in response to the stress event, with a similar trend in males and females. Hormone levels were back to baseline values in both groups a few days after transfer and relocation. The presence of known social partners could be one of the factors that possibly facilitated the adaptation process.

Transcriptomic regulations in oligodendroglial and microglial cells related to brain damage following fetal growth restriction.

Fetal growth restriction (FGR) is a major complication of human pregnancy, frequently resulting from placental vascular diseases and prenatal malnutrition, and is associated with adverse neurocognitive outcomes throughout life. However, the mechanisms linking poor fetal growth and neurocognitive impairment are unclear. Here, we aimed to correlate changes in gene expression induced by FGR in rats and abnormal cerebral white matter maturation, brain microstructure, and cortical connectivity in vivo. We investigated a model of FGR induced by low-protein-diet malnutrition between embryonic day 0 and birth using an interdisciplinary approach combining advanced brain imaging, in vivo connectivity, microarray analysis of sorted oligodendroglial and microglial cells and histology. We show that myelination and brain function are both significantly altered in our model of FGR. These alterations, detected first in the white matter on magnetic resonance imaging significantly reduced cortical connectivity as assessed by ultrafast ultrasound imaging. Fetal growth retardation was found associated with white matter dysmaturation as shown by the immunohistochemical profiles and microarrays analyses. Strikingly, transcriptomic and gene network analyses reveal not only a myelination deficit in growth-restricted pups, but also the extensive deregulation of genes controlling neuroinflammation and the cell cycle in both oligodendrocytes and microglia. Our findings shed new light on the cellular and gene regulatory mechanisms mediating brain structural and functional defects in malnutrition-induced FGR, and suggest, for the first time, a neuroinflammatory basis for the poor neurocognitive outcome observed in growth-restricted human infants. GLIA 2016;64:2306-2320.

Glial response to 17ß-estradiol in neonatal rats with excitotoxic brain injury.

White-matter injury is the most common cause of the adverse neurodevelopmental outcomes observed in preterm infants. Only few options exist to prevent perinatal brain injury associated to preterm delivery. 17ß-estradiol (E2) is the predominant estrogen in circulation and has been shown to be neuroprotective in vitro and in vivo. However, while E2 has been found to modulate inflammation in adult models of brain damage, how estrogens influence glial cells response in the developing brain needs further investigations. Using a model of ibotenate-induced brain injury, we have refined the effects of E2 in the developing brain. E2 provides significant neuroprotection both in the cortical plate and the white matter in neonatal rats subjected to excitotoxic insult mimicking white matter and cortical damages frequently observed in very preterm infants. E2 promotes significant changes in microglial phenotypes balance in response to brain injury and the acceleration of oligodendrocyte maturation. Maturational effects of E2 on myelination process were observed both in vivo and in vitro. Altogether, these data demonstrate that response of glial cells to E2 could be responsible for its neuroprotective properties in neonatal excitotoxic brain injury.

Evidence for an imbalance between tau O-GlcNAcylation and phosphorylation in the hippocampus of a mouse model of Alzheimer's disease.

Intracellular accumulation of hyperphosphorylated tau protein is linked to neuronal degeneration in Alzheimer's disease (AD). Mounting evidence suggests that tau phosphorylation and O-N-acetylglucosamine glycosylation (O-GlcNAcylation) are mutually exclusive post-translational modifications. O-GlcNAcylation depends on 3-5% of intracellular glucose that enters the hexosamine biosynthetic pathway. To our knowledge, the existence of an imbalance between tau phosphorylation and O-GlcNAcylation has not been reported in animal models of AD, as yet. Here, we used triple transgenic (3xTg-AD) mice at 12 months, an age at which hyperphosphorylated tau is already detected and associated with cognitive decline. In these mice, we showed that tau was hyperphosphorylated on both Ser396 and Thr205 in the hippocampus, and to a lower extent and exclusively on Thr205 in the frontal cortex. Tau O-GlcNAcylation, assessed in tau immunoprecipitates, was substantially reduced in the hippocampus of 3xTg-AD mice, with no changes in the frontal cortex or in the cerebellum. No changes in the expression of the three major enzymes involved in O-GlcNAcylation, i.e., glutamine fructose-6-phosphate amidotransferase, O-linked ß-N-acetylglucosamine transferase, and O-GlcNAc hydrolase were found in the hippocampus of 3xTg-AD mice. These data demonstrate that an imbalance between tau phosphorylation and O-GlcNAcylation exists in AD mice, and strengthens the hypothesis that O-GlcNAcylation might be targeted by disease modifying drugs in AD.