Centre de Recherche de l'Institut de Démographie de l'Université Paris 1

Inflammation is increasingly recognized as being of both physiological and pathological importance in the immature brain. The rationale of this review is to present an update on this topic with focus on long-term consequences of inflammation during childhood and in adults. The immature brain can be exposed to inflammation in connection with viral or bacterial infection during pregnancy or as a result of sterile central nervous system (CNS) insults. Through efficient anti-inflammatory and reparative processes, inflammation may resolve without any harmful effects on the brain. Alternatively, inflammation contributes to injury or enhances CNS vulnerability. Acute inflammation can also be shifted to a chronic inflammatory state and/or adversely affect brain development. Hypothetically, microglia are the main immunocompetent cells in the immature CNS, and depending on the stimulus, molecular context, and timing, these cells will acquire various phenotypes, which will be critical regarding the CNS consequences of inflammation. Inflammation has long-term consequences and could speculatively modify the risk of a variety of neurological disorders, including cerebral palsy, autism spectrum disorders, schizophrenia, multiple sclerosis, cognitive impairment, and Parkinson disease. So far, the picture is incomplete, and data mostly experimental. Further studies are required to strengthen the associations in humans and to determine whether novel therapeutic interventions during the perinatal period can influence the occurrence of neurological disease later in life.

OBJECTIVE: Perinatal inflammation is a major risk factor for neurological deficits in preterm infants. Several experimental studies have shown that systemic inflammation can alter the programming of the developing brain. However, these studies do not offer detailed pathophysiological mechanisms, and they rely on relatively severe infectious or inflammatory stimuli that most likely do not reflect the levels of systemic inflammation observed in many human preterm infants. The goal of the present study was to test the hypothesis that moderate systemic inflammation is sufficient to alter white matter development. METHODS: Newborn mice received twice-daily intraperitoneal injections of interleukin-1β (IL-1β) over 5 days and were studied for myelination, oligodendrogenesis, and behavior and with magnetic resonance imaging (MRI). RESULTS: Mice exposed to IL-1β had a long-lasting myelination defect that was characterized by an increased number of nonmyelinated axons. They also displayed a reduction of the diameter of the myelinated axons. In addition, IL-1β induced a significant reduction of the density of myelinating oligodendrocytes accompanied by an increased density of oligodendrocyte progenitors, suggesting a partial blockade in the oligodendrocyte maturation process. Accordingly, IL-1β disrupted the coordinated expression of several transcription factors known to control oligodendrocyte maturation. These cellular and molecular abnormalities were correlated with a reduced white matter fractional anisotropy on diffusion tensor imaging and with memory deficits. INTERPRETATION: Moderate perinatal systemic inflammation alters the developmental program of the white matter. This insult induces a long-lasting myelination deficit accompanied by cognitive defects and MRI abnormalities, further supporting the clinical relevance of the present data.

Abstract Daunorubicin induces complete remissions in about 50% of patients with acute promyelocytic leukemia. The median duration of these remission is 26 mo. Failures are mainly due to hemorrhages as a result of disseminated intravascular coagulation during the first 5 days (25%) or due to sepsis during the second and third week (25%). Long-term survivals are more frequent than in the other acute granulocytic leukemias.

Amoeboid microglial subpopulations visualized by antibodies against ionized calcium-binding adapter molecule 1, CD68, and CD45 enter the forebrain starting at 4.5 postovulatory or gestational weeks (gw). They penetrate the telencephalon and diencephalon via the meninges, choroid plexus, and ventricular zone. Early colonization by amoeboid microglia-macrophages is first restricted to the white matter, where these cells migrate and accumulate in patches at the junctions of white-matter pathways, such as the three junctions that the internal capsule makes with the thalamocortical projection, external capsule and cerebral peduncle, respectively. In the cerebral cortex anlage, migration is mainly radial and tangential towards the immature white matter, subplate layer, and cortical plate, whereas pial cells populate the prospective layer I. A second wave of microglial cells penetrates the brain via the vascular route at about 12-13 gw and remains confined to the white matter. Two main findings deserve emphasis. First, microglia accumulate at 10-12 gw at the cortical plate-subplate junction, where the first synapses are detected. Second, microglia accumulate in restricted laminar bands, most notably around 19-30 gw, at the axonal crossroads in the white matter (semiovale centre) rostrally, extending caudally in the immature white matter to the visual radiations. This accumulation of proliferating microglia is located at the site of white-matter injury in premature neonates. The spatiotemporal organization of microglia in the immature white and grey matter suggests that these cells may play active roles in developmental processes such as axonal guidance, synaptogenesis, and neurodevelopmental apoptosis as well as in injuries to the developing brain, in particular in the periventricular white-matter injury of preterm infants.

OBJECTIVE: Activated microglia play a central role in the inflammatory and excitotoxic component of various acute and chronic neurological disorders. However, the mechanisms leading to their activation in the latter context are poorly understood, particularly the involvement of N-methyl-D-aspartate receptors (NMDARs), which are critical for excitotoxicity in neurons. We hypothesized that microglia express functional NMDARs and that their activation would trigger neuronal cell death in the brain by modulating inflammation. METHODS AND RESULTS: We demonstrate that microglia express NMDARs in the murine and human central nervous system and that these receptors are functional in vitro. We show that NMDAR stimulation triggers microglia activation in vitro and secretion of factors that induce cell death of cortical neurons. These damaged neurons are further shown to activate microglial NMDARs and trigger a release of neurotoxic factors from microglia in vitro, indicating that microglia can signal back to neurons and possibly induce, aggravate, and/or maintain neurologic disease. Neuronal cell death was significantly reduced through pharmacological inhibition or genetically induced loss of function of the microglial NMDARs. We generated Nr1 LoxP(+/+) LysM Cre(+/-) mice lacking the NMDAR subunit NR1 in cells of the myeloid lineage. In this model, we further demonstrate that a loss of function of the essential NMDAR subunit NR1 protects from excitotoxic neuronal cell death in vivo and from traumatic brain injury. INTERPRETATION: Our findings link inflammation and excitotoxicity in a potential vicious circle and indicate that an activation of the microglial NMDARs plays a pivotal role in neuronal cell death in the perinatal and adult brain.

BACKGROUND: During the first weeks of hospitalization, premature babies and their parents encounter difficulties in establishing early bonds and interactions. Only a few studies have explored what caregivers can do to meet parents' needs in relation to these interactions and help optimize them. This study sought to explore parents' perception of these first interactions and to identify the actions of caregivers that help or hinder its development. METHODS: Prospective study, qualitative discourse analysis of 60 face-to-face interviews conducted with 30 mothers and 30 fathers of infants born before 32 weeks of gestation (mean ± SD: 27 ± 2 weeks of gestational age), during their child's stay in one out of three NICUs in France. Interviews explored parental experience, from before birth up to the first month of life. RESULTS: Data analysis uncovered two main themes, which were independent of parents' geographical or cultural origin but differed between mothers and fathers. First, fathers described the bond with their child as composed more of words and looks and involving distance, while mothers experienced the bond more physically. Secondly, two aspects of the caregivers' influence were decisive: nurses' caring attitude towards baby and parents, and their communication with parents, which reduced stress and made interactions with the baby possible. This communication appeared to be the locus of a supportive and fulfilling encounter between parents and caregivers that reinforced parents' perception of a developing bond. CONCLUSIONS: At birth and during the first weeks in the NICU, the creation of a bond between mothers and fathers and their premature baby is rooted in their relationship with the caregivers. Nurses' caring attitude and regular communication adapted to specific needs are perceived by parents as necessary preconditions for parents' interaction and development of a bond with their baby. These results might allow NICU staff to provide better support to parents and facilitate the emergence of a feeling of parenthood.

Disorders of the developing brain represent a major health problem. The neurological manifestations of brain lesions can range from severe clinical deficits to more subtle neurological signs or behavioral problems and learning disabilities, which often become evident many years after the initial damage. These long-term sequelae are due at least in part to central nervous system immaturity at the time of the insult. The blood-brain barrier (BBB) protects the brain and maintains homeostasis. BBB alterations are observed during both acute and chronic brain insults. After an insult, excitatory amino acid neurotransmitters are released, causing reactive oxygen species (ROS)-dependent changes in BBB permeability that allow immune cells to enter and stimulate an inflammatory response. The cytokines, chemokines and other molecules released as well as peripheral and local immune cells can activate an inflammatory cascade in the brain, leading to secondary neurodegeneration that can continue for months or even years and finally contribute to post-insult neuronal deficits. The role of the BBB in perinatal disorders is poorly understood. The inflammatory response, which can be either acute (e.g., perinatal stroke, traumatic brain injury) or chronic (e.g., perinatal infectious diseases) actively modulates the pathophysiological processes underlying brain injury. We present an overview of current knowledge about BBB dysfunction in the developing brain during acute and chronic insults, along with clinical and experimental data.

Disabilities after brain injury in very preterm infants have mainly been attributed to noncystic periventricular white matter injury (PWMI). We analyzed spatiotemporal patterns of PWMI in the brains of 18 very preterm infants (25-29 postconceptional weeks [pcw]), 7 preterm infants (30-34 pcw), and 10 preterm controls without PWMI. In very preterm infants, we examined PWMI in detail in 2 axonal crossroad areas in the frontal lobe: C1 (lateral to the lateral angle of the anterior horn of the lateral ventricle, at the exit of the internal capsule radiations) and C2 (above the corpus callosum and dorsal angle of the anterior horn). These brains had greater microglia-macrophage densities and activation but lesser astroglial reaction (glial fibrillary acidic protein and monocarboxylate transporter 1 expression) than in preterm cases with PWMI. In preterm infants, scattered necrotic foci were rimmed by axonal spheroids and ionized calcium binding adaptor molecule 1-positive macrophages. Diffuse lesions near these foci consisted primarily of hypertrophic and reactive astrocytes associated with fewer microglia. No differences in Olig2-positive preoligodendrocytes between noncystic PWMI and control cases were found. These data show that the growing axonal crossroad areas are highly vulnerable to PWMI in very preterm infants and highlight differences in glial activation patterns between very preterm and preterm infants.

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/β-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

Cerebral palsy is a major health problem caused by brain damage during pregnancy, delivery, or the immediate postnatal period. Perinatal stroke, intraventricular hemorrhage, and asphyxia are the most common causes of neonatal brain damage. Periventricular white matter damage (periventricular leukomalacia) is the predominant form in premature infants and the most common antecedent of cerebral palsy. Stem cell treatment has proven effective in restoring injured organs and tissues in animal models. The potential of stem cells for self-renewal and differentiation translates into substantial neuroprotection and neuroregeneration in the animal brain, with minimal risks of rejection and side effects. Stem cell treatments described to date have used neural stem cells, embryonic stem cells, mesenchymal stem cells, umbilical cord stem cells, and induced pluripotent stem cells. Most of these treatments are still experimental. In this review, we focus on the efficacy of stem cell therapy in animal models of cerebral palsy, and discuss potential implications for current and future clinical trials.

Preterm birth places infants in an adverse environment that leads to abnormal brain development and cerebral injury through a poorly understood mechanism known to involve neuroinflammation. In this study, we integrate human and mouse molecular and neuroimaging data to investigate the role of microglia in preterm white matter damage. Using a mouse model where encephalopathy of prematurity is induced by systemic interleukin-1β administration, we undertake gene network analysis of the microglial transcriptomic response to injury, extend this by analysis of protein-protein interactions, transcription factors and human brain gene expression, and translate findings to living infants using imaging genomics. We show that DLG4 (PSD95) protein is synthesised by microglia in immature mouse and human, developmentally regulated, and modulated by inflammation; DLG4 is a hub protein in the microglial inflammatory response; and genetic variation in DLG4 is associated with structural differences in the preterm infant brain. DLG4 is thus apparently involved in brain development and impacts inter-individual susceptibility to injury after preterm birth.Inflammation mediated by microglia plays a key role in brain injury associated with preterm birth, but little is known about the microglial response in preterm infants. Here, the authors integrate molecular and imaging data from animal models and preterm infants, and find that microglial expression of DLG4 plays a role.

Perinatal insults are a leading cause of infant mortality and amongst survivors are frequently associated with neurocognitive impairment, cerebral palsy (CP), and seizure disorders. The events leading to perinatal brain injury are multifactorial. This review describes how one subinjurious factor affecting the brain sensitizes it to a second injurious factor, causing an exacerbated injurious cascade. We will review the clinical and experimental evidence, including observations of high rates of maternal and fetal infections in term-born infants with neonatal encephalopathy and cerebral palsy. In addition, we will discuss preclinical evidence for the sensitizing effects of inflammation on injuries, such as hypoxia-ischaemia, our current understanding of the mechanisms underpinning the sensitization process, and the possibility for neuroprotection.

A rapid growth in human cerebellar development occurs in the third trimester, which is impeded by preterm delivery. The goal of this study was to characterize the impact of preterm delivery on the developmental program of the human cerebellum. Still born infants, which meant that all development up to that age had taken place in-utero, were age paired with preterm delivery infants, who had survived in an ex-utero environment, which meant that their development had also taken place outside the uterus. The two groups were assessed on quantitative measures that included molecular markers of granule neuron, purkinje neuron and bergmann glia differentiation, as well as the expression of the sonic hedgehog signaling pathway, that is important for cerebellar growth. We report that premature birth and development in an ex-utero environment leads to a significant decrease in the thickness and an increase in the packing density of the cells within the external granular layer and the inner granular layer well, as a reduction in the density of bergmann glial fibres. In addition, this also leads to a reduced expression of sonic hedgehog in the purkinje layer. We conclude that the developmental program of the cerebellum is specifically modified by events that follow preterm delivery.

The cognitive and behavioural deficits caused by traumatic brain injury (TBI) to the immature brain are more severe and persistent than TBI in the mature brain. Understanding this developmental sensitivity is critical as children under four years of age sustain TBI more frequently than any other age group. Microglia (MG), resident immune cells of the brain that mediate neuroinflammation, are activated following TBI in the immature brain. However, the type and temporal profile of this activation and the consequences of altering it are still largely unknown. In a mouse model of closed head weight drop paediatric brain trauma, we characterized i) the temporal course of total cortical neuroinflammation and the phenotype of ex vivo isolated CD11B-positive microglia/macrophage (MG/MΦ) using a battery of 32 markers, and ii) neuropathological outcome 1 and 5days post-injury. We also assessed the effects of targeting MG/MΦ activation directly, using minocycline a prototypical microglial activation antagonist, on these processes and outcome. TBI induced a moderate increase in both pro- and anti-inflammatory cytokines/chemokines in the ipsilateral hemisphere. Isolated cortical MG/MΦ expressed increased levels of markers of endogenous reparatory/regenerative and immunomodulatory phenotypes compared with shams. Blocking MG/MΦ activation with minocycline at the time of injury and 1 and 2days post-injury had only transient protective effects, reducing ventricular dilatation and cell death 1day post-injury but having no effect on injury severity at 5days. This study demonstrates that, unlike in adults, the role of MG/MΦ in injury mechanisms following TBI in the immature brain may not be negative. An improved understanding of MG/MΦ function in paediatric TBI could support translational efforts to design therapeutic interventions.

Trophoblastic cell-cell fusion is an essential event required during human placental development. Several membrane proteins have been described to be directly involved in this process, including connexin 43 (Cx43), syncytin 1 (Herv-W env), and syncytin 2 (Herv-FRD env glycoprotein). Recently, zona occludens (ZO) proteins (peripheral membrane proteins associated with tight junctions, adherens junctions, and gap junctions) were shown to be involved in mouse placental development. Moreover, zona occludens 1 (ZO-1) was localized mainly at the intercellular boundaries between human trophoblastic cells. Therefore the role of ZO-1 in the dynamic process of human trophoblastic cell-cell fusion was investigated using primary trophoblastic cells in culture. In vitro as in situ, ZO-1 was localized mainly at the intercellular boundaries between trophoblastic cells where its expression substantially decreased during differentiation and during fusion. At the same time, Cx43 was localized at the interface of trophoblastic cells and its expression increased during differentiation. To determine a functional role for ZO-1 during trophoblast differentiation, small interfering RNA (siRNA) was used to knock down ZO-1 expression. Cytotrophoblasts treated with ZO-1 siRNA fused poorly, but interestingly, decreased Cx43 expression without altering the functionality of trophoblastic cell-cell communication as measured by relative permeability time constant determined using gap-FRAP experiments. Because kinetics of Cx43 and ZO-1 proteins show a mirror image, a potential association of these two proteins was investigated. By using coimmunoprecipitation experiments, a physical interaction between ZO-1 and Cx43 was demonstrated. These results demonstrate that a decrease in ZO-1 expression reduces human trophoblast cell-cell fusion and differentiation.

Cerebellar injury is increasingly recognized through advanced neonatal brain imaging as a complication of premature birth. Survivors of preterm birth demonstrate a constellation of long-term neurodevelopmental deficits, many of which are potentially referable to cerebellar injury, including impaired motor functions such as fine motor incoordination, impaired motor sequencing and also cognitive, behavioral dysfunction among older patients. This paper reviews the morphogenesis and histogenesis of the human and rodent developing cerebellum, and its more frequent injuries in preterm. Most cerebellar lesions are cerebellar hemorrhage and infarction usually leading to cerebellar abnormalities and/or atrophy, but the exact pathogenesis of lesions of the cerebellum is unknown. The different mechanisms involved have been investigated with animal models and are primarily hypoxia, ischemia, infection, and inflammation Exposure to drugs and undernutrition can also induce cerebellar abnormalities. Different models are detailed to analyze these various disturbances of cerebellar development around birth.

Intrauterine growth restriction (IUGR) is a complex global healthcare issue. Concerted research and clinical efforts have improved our knowledge of the neurodevelopmental sequelae of IUGR which has raised the profile of this complex problem. Nevertheless, there is still a lack of therapies to prevent the substantial rates of fetal demise or the constellation of permanent neurological deficits that arise from IUGR. The purpose of this article is to highlight the clinical and translational gaps in our knowledge that hamper our collective efforts to improve the neurological sequelae of IUGR. Also, we draw attention to cutting-edge tools and techniques that can provide novel insights into this disorder, and technologies that offer the potential for better drug design and delivery. We cover topics including: how we can improve our use of crib-side monitoring options, what we still need to know about inflammation in IUGR, the necessity for more human post-mortem studies, lessons from improved integrated histology-imaging analyses regarding the cell-specific nature of magnetic resonance imaging (MRI) signals, options to improve risk stratification with genomic analysis, and treatments mediated by nanoparticle delivery which are designed to modify specific cell functions.

Epidemiological studies have shown a strong association between perinatal infection/inflammation and brain damage in preterm infants and/or neurological handicap in survivors. Experimental studies have shown a causal effect of infection/inflammation on perinatal brain damage. Infection including inflammatory factors can disrupt programmes of brain development and, in particular, induce death and/or blockade of oligodendrocyte maturation, leading to myelin defects. Alternatively, in the so-called multiple-hit hypothesis, infection/inflammation can act as predisposing factors, making the brain more susceptible to a second stress (sensitization process), such as hypoxic-ischaemic or excitotoxic insults. Epidemiological data also suggest that perinatal exposure to inflammatory factors could predispose to long-term diseases including psychiatric disorders.

BACKGROUND: Alveolarization requires coordinated extracellular matrix remodeling, a process in which matrix metalloproteinases (MMPs) play an important role. We postulated that polymorphisms in MMP genes might affect MMP function in preterm lungs and thus influence the risk of bronchopulmonary dysplasia (BPD). METHODS AND FINDINGS: Two hundred and eighty-four consecutive neonates with a gestational age of <28 weeks were included in this prospective study. Forty-five neonates developed BPD. Nine single-nucleotide polymorphisms (SNPs) were sought in the MMP2, MMP14 and MMP16 genes. After adjustment for birth weight and ethnic origin, the TT genotype of MMP16 C/T (rs2664352) and the GG genotype of MMP16 A/G (rs2664349) were found to protect from BPD. These genotypes were also associated with a smaller active fraction of MMP2 and with a 3-fold-lower MMP16 protein level in tracheal aspirates collected within 3 days after birth. Further evaluation of MMP16 expression during the course of normal human and rat lung development showed relatively low expression during the canalicular and saccular stages and a clear increase in both mRNA and protein levels during the alveolar stage. In two newborn rat models of arrested alveolarization the lung MMP16 mRNA level was less than 50% of normal. CONCLUSIONS: MMP16 may be involved in the development of lung alveoli. MMP16 polymorphisms appear to influence not only the pulmonary expression and function of MMP16 but also the risk of BPD in premature infants.

Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP- and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.