[Neonatal medicine, past and present]. / Néonatologie: passé et present.

This review deals with early neonatal medicine and its rapid development as a medical specialty, starting with the birth of neonatology in the early 19th century. Shaffer first used the term neonatology in 1963 to cover neonatal disorders and their treatment. Between the early 19th century and the 1950s, neonatal care was ensured by obstetricians, whose main goal was to reduce neonatal mortality. After the second world war, and especially the 1960s, the development of neonatal physiology and pathophysiology provided insights into neonatal diseases and their treatment, including respiratory distress, jaundice, malnutrition, and prevention of respiratory distress and brain complications, etc. Currently, neonatal mortality, regardless of birth weight, is below 2/1000, and the survival rate of premature infants, regardless of gestational age and birth weight, exceeds 85%. This represents a resounding success, despite the associated costs, ethical issues, and inevitable morbidity.

Non-injurious neonatal hypoxia confers resistance to brain senescence in aged male rats.

Whereas brief acute or intermittent episodes of hypoxia have been shown to exert a protective role in the central nervous system and to stimulate neurogenesis, other studies suggest that early hypoxia may constitute a risk factor that influences the future development of mental disorders. We therefore investigated the effects of a neonatal "conditioning-like" hypoxia (100% N2, 5 min) on the brain and the cognitive outcomes of rats until 720 days of age (physiologic senescence). We confirmed that such a short hypoxia led to brain neurogenesis within the ensuing weeks, along with reduced apoptosis in the hippocampus involving activation of Erk1/2 and repression of p38 and death-associated protein (DAP) kinase. At 21 days of age, increased thicknesses and cell densities were recorded in various subregions, with strong synapsin activation. During aging, previous exposure to neonatal hypoxia was associated with enhanced memory retrieval scores specifically in males, better preservation of their brain integrity than controls, reduced age-related apoptosis, larger hippocampal cell layers, and higher expression of glutamatergic and GABAergic markers. These changes were accompanied with a marked expression of synapsin proteins, mainly of their phosphorylated active forms which constitute major players of synapse function and plasticity, and with increases of their key regulators, i.e. Erk1/2, the transcription factor EGR-1/Zif-268 and Src kinase. Moreover, the significantly higher interactions between PSD-95 scaffolding protein and NMDA receptors measured in the hippocampus of 720-day-old male animals strengthen the conclusion of increased synaptic functional activity and plasticity associated with neonatal hypoxia. Thus, early non-injurious hypoxia may trigger beneficial long term effects conferring higher resistance to senescence in aged male rats, with a better preservation of cognitive functions.

[Folates and fetal programming: role of epigenetics and epigenomics]. / Folates et programmation foetale: rôle des mécanismes nutrigénomiques et épigénomiques.

Folates are needed for synthesis of methionine, the precursor of S-adenosyl methionine (SAM). They play therefore a key role in nutrition and epigenomics by fluxing monocarbons towards synthesis or methylation of DNA and RNA, and methylation of gene transregulators, respectively. The deficiency produces intrauterine growth retardation and birth dejects. Folate deficiency deregulates epigenomic mechanisms related to fetal programming through decreased cellular availability of SAM. Epigenetic mechanisms of folate deficiency are illustrated by inheritance of coat colour of agouti mice model and altered expression of Igf2/H19 imprinting genes. Dietary exposure to fumonisin FB1 acts synergistically with folate deficiency on alterations of heterochromatin assembly. Deficiency in folate and vitamin B12 produces impaired fatty acid oxidation in liver and heart through imbalanced methylation and acetylation of PGC1-alpha and decreased expression of SIRT1, and long-lasting cognitive disabilities through impaired hippocampal cell proliferation, differentiation and plasticity and atrophy of hippocampal CA1. Deciphering these mechanisms will help understand the discordances between experimental models and population studies on folate supplementation.

Conditioning-like brief neonatal hypoxia improves cognitive function and brain tissue properties with marked gender dimorphism in adult rats.

Although recent studies have documented compensatory generation of neurons in adult brains in response to various insults, a noninjurious short episode of hypoxia in rat neonates has been shown to trigger neurogenesis within the ensuing weeks, without apparent brain lesions. Very little is known of the long-term consequences. We therefore investigated the effects of such a conditioning-like hypoxia (100% N(2), 5 min) on the brain and the cognitive outcomes of rats at 40 to 100 days of age. Control and posthypoxic rats developed similar learning capacities over postnatal days 14 to 18, but hypoxia was associated with enhanced scores in a test used to evaluate memory retrieval between 40 and 100 days. A striking sexual dimorphism was observed, with an earlier functional gain observed in female (40 days) compared with male (100 days) rats; gains were associated with matching structural changes in areas involved in cognition, including the hippocampus and frontal cortex. Therefore, it is proposed that brief neonatal hypoxia may exert long-term beneficial effects through neurogenesis stimulation.

[Infants of drug-addicted mothers: pitfalls of replacement therapy]. / Enfants de mères toxicomanes: les aléas de la substitution.

Maternal drug addiction can cause problems for the fetus and the newborn, and hamper long-term development. The prevalence of drug addiction during pregnancy varies from 1 % to more than 10 % depending on the country and the maternity unit. Management of these mothers can be further complicated by medical, social and psychological problems. Compared to methadone, heroin replacement therapy with buprenorphine provides better stabilization of the mother and causes fewer withdrawal symptoms in the newborn. Despite numerous publications on the effects of this partly preventive medication, data on buprenorphine pharmacology at birth are scarce. In this study, 20 newborns of mothers using oral buprenorphine were observed until the end of the withdrawal syndrome, when present. Buprenorphine plasma levels were determined with HPLC and mass spectrometry in the mother at delivery and in the newborn at birth (cord blood), 24 and 48 hours. Fifteen newborns were born at term (mean +/- SD birth weight 3029 +/- 273 g), and the other five between 32 and 36 weeks. All Apgar scores were > or =7. Withdrawal symptoms were observed in 8 of the 15 infants born to mothers taking buprenorphine alone, and lasted between 5 and 35 days. The newborns were classified in three groups. Groups I (N8) and II (N7) comprised newborns with and without withdrawal symptoms, respectively. In group III (N5), the mothers were polyintoxicated (as shown by urinary drug or neurotropic substance screening) and the newborns were symptomatic for 1 to 69 days. Buprenorphine plasma levels in the mothers ranged from 0 to 2.9 microg/L, suggesting large differences in adherence. At birth there was no significant difference in the mean plasma buprenorphine level between newborns with and without withdrawal symptoms; the respective values were 0.7 (0.4-1.3) and 0.5 (0-0.6) microg/L. In asymptomatic newborns (group II), buprenorphine was no longer detectable at 48 h, whereas in symptomatic newborns (group I), the mean level rose from 0.7 microg/l at birth to 1.5 microg/L at 48 h (+114 %). In the absence of breastfeeding, this increase appears to be related to tissue release of this strongly lipophilic compound. The difference in plasma buprenorphine kinetics between groups I and II might be explained by genetic polymorphism of drug-metabolizing enzymes. The paradoxically high plasma buprenorphine levels at 48 hours in infants with withdrawal symptoms are intriguing. One possibility is that the mothers missed one or several doses of buprenorphine around the time of delivery, in the same way that smoking mothers tend to cut down during the last days of their pregnancy. If buprenorphine plasma levels at birth appear to reflect maternal adherence, cord blood levels do not predict the risk of a withdrawal syndrome. In contrast, the level at 48 h might help to discriminate between high- and low-risk newborns. Pregnant women on opiate replacement therapy must be delivered in maternity units with adequate neonatal facilities.

[Cell death and neurogenesis after hypoxia: a brain repair mechanism in the developing rat?]. / Mort cellulaire et neurogenèse post-hypoxiques: un mécanisme de réparation cérébrale chez le rat en développement?

Perinatal brain damage following hypoxia-ischemia has long been considered irreversible, but recent rodent studies show that various insults can induce de novo neurogenesis in the adult brain. Here we examined whether acute hypoxia could trigger neurogenesis in the developing rat brain. In vitro, we examined the impact of transient hypoxia on cultured embryonic rat neurons. In vivo, we monitored the time course of brain damage in the CA1 layer of the hippocampus of one-day-old rats after exposure to hypoxia. The extent of cell loss and regeneration was evaluated after staining with DAPI. Newly generated cells were characterized in the subventricular zone by immunohistochemistry, 20 days post-exposure. The viability of cultured neurons was reduced by 36 % when measured 96 h after 6 h of hypoxia, and a significant number of cell nuclei showed apoptotic features. In contrast, cell numbers increased by 14 % 96 h after 3 h of hypoxia. The Bax/Bcl2 ratio tended to increase after 6 h of hypoxia and to decrease after 3 h of hypoxia. In the CA1 subfield of the hippocampus, hypoxia reduced the total number of cells by 27 % on day 6-7 post-reoxygenation, and histopathological hallmarks of apoptosis were observed This cell deficit was followed by gradual recovery, starting on day 20, suggesting a repair mechanism. BrdU incorporation in the subventricular zone showed an accumulation of proliferating cells expressing the neuronal marker NeuroD. These data show that post-hypoxic neurogenesis can occur during development.

Apoptosis and neurogenesis after transient hypoxia in the developing rat brain.

Perinatal brain damage following a hypoxic-ischemic episode has been considered for a long time as an irreversible phenomenon. However, recent studies have shown that various insults may induce de novo neurogenesis in the adult rodent brain. The present study tested the hypothesis that acute hypoxia may trigger neurogenesis in the developing brain. In vitro, the influence of transient hypoxia was analyzed on the outcome of embryonic rat neurons in culture. In vivo, the temporal profile of brain damage was monitored at the level of the CA1 layer of the hippocampus after the exposure to hypoxia of 1-day-old rats. The extent of cell loss and regeneration was evaluated after staining with DAPI. The characterization of newly generated cells was performed in the subventricular zone at 20 days postexposure by immunohistochemistry. Following hypoxia for 6 hours, neuronal viability in the culture dishes was reduced by 36% at 96 hours, with a significant number of cell nuclei showing apoptosis features. In contrast, a 3-hour hypoxia apparently did not damage cultured neurons whose number increased by 14%. The Bax/Bcl-2 ratio tended to increase after 6-hour hypoxia and to decrease after 3-hour hypoxia. In vivo, hypoxia induced cell damage in the CA1 subfield of the hippocampus, where the total number of cells was reduced by 27% at days 6-7 postreoxygenation, with histopathological hallmarks of apoptosis. This cell deficit was followed by a gradual recovery observable from day 20, suggesting a repair mechanism. Brain incorporation of BrdU in the subventricular zone revealed an accumulation of proliferating cells expressing the neuronal marker NeuroD. The present data demonstrate that a posthypoxic neurogenesis does occur during development and may account for brain protection.

Neonatal hypoxia triggers transient apoptosis followed by neurogenesis in the rat CA1 hippocampus.

Continuous generation of new neurons has been demonstrated in the adult mammalian brain, and this process was shown to be stimulated by various pathologic conditions, including cerebral ischemia. Because brain oxygen deprivation is particularly frequent in neonates and represents the primary event of asphyxia, we analyzed long-term consequences of transient hypoxia in the newborn rat. Within 24 h after birth, animals were exposed to 100% N(2) for 20 min at 36 degrees C, and temporal changes in the vulnerable CA1 hippocampus were monitored. Cell density measurements revealed delayed cell death in the pyramidal cell layer reflecting apoptosis, as shown by characteristic nuclear morphology and expression levels of Bcl-2, Bax, and caspase-3. Neuronal loss was confirmed by reduced density of neuron-specific enolase (NSE)-labeled cells, and peaked by 1 wk post insult, to reach 27% of total cells. A gradual recovery then occurred, and no significant difference in cell density could be detected between controls and hypoxic rats at postnatal d 21. Repeated injections of bromodeoxyuridine (50 mg/kg) showed that newly divided cells expressing neuronal markers increased by 225% in the germinative subventricular zone, and they tended to migrate along the posterior periventricle toward the hippocampus. Therefore, transient hypoxia in the newborn rat triggered apoptosis in the CA1 hippocampus followed by increased neurogenesis and apparent anatomical recovery, suggesting that the developing brain may have a high capacity for self-repair.

Histopathological alterations and functional brain deficits after transient hypoxia in the newborn rat pup: a long term follow-up.

To assess temporal brain deficits consecutive to severe birth hypoxia, newborn rats were exposed for 20 min to 100% N2. This treatment induced a long-term growth retardation and a delayed, but only transient, neuronal loss (approximately 25%) in the CA1 hippocampus and parietal cortex, starting from 3 days and peaking at 6 days post-hypoxia. The expression profiles of various apoptosis-regulating proteins (including Bcl-2, Bax, p53 and caspase-3) were well correlated to the alterations of nuclear morphology depicted by 4,6-diamidino-2-phenylindole (DAPI). Whereas they confirmed a gradual histological recovery, specific DNA fragmentation patterns suggested that birth hypoxia may transiently reactivate the developmental programme of neuronal elimination. Although they successfully achieved various behavioral tests such as the righting reflex, negative geotaxis, locomotor coordination, and the eight-arm maze tasks, both developing and adult hypoxic rats were repeatedly slower than controls, suggesting that birth hypoxia is associated to moderate but persistent impairments of functional capacities.

Differential neuronal fates in the CA1 hippocampus after hypoxia in newborn and 7-day-old rats: effects of pre-treatment with MK-801.

The brain displays an age-dependent sensitivity to ischemic insults. However, the consequences of oxygen deprivation per se in the developing brain remain unclear, and the role of glutamate excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is controversial. To gain a better understanding of the mechanisms involved in the cerebral response to severe hypoxia, cell damage was temporally monitored in the CA1 hippocampus of rat pups transiently exposed to in vivo hypoxia (100% N2) at either 24 h or 7 days of age. Also, the influence of a pre-treatment with the NMDA receptor antagonist MK-801 (5 mg/kg, i.p.) was examined. At both ages, morphometric analyses and cell counts showed hypoxia-induced significant neuronal loss (30-35%) in the pyramidal layer, with injury appearing more rapidly in rats exposed at 7 days. Morphological alterations of 4,6-diamidino-2-phenylindole (DAPI)-labeled nuclei, DNA fragmentation patterns on agarose gels, as well as expression profiles of the apoptosis-related regulatory proteins Bax and Bcl-2 showed that apoptosis was prevalent in younger animals, whereas only necrosis was detected in hippocampi of rats treated at 7 days. Moreover, pre-treatment with MK-801 was ineffective in protecting hippocampal neurons from hypoxic injury in newborn rats, but significantly reduced necrosis in older subjects. These data confirm that hypoxia alone may trigger neuronal death in vivo, and the type of cell death is strongly influenced by the degree of brain maturity. Finally, NMDA receptors are not involved in the apoptotic consequences of hypoxia in the newborn rat brain, but they were found to mediate necrosis at 7 days of age.

Combined effects of bilirubin and hypoxia on cultured neurons from the developing rat forebrain.

Hyperbilirubinemia and hypoxia are common causes of brain injury in the newborn. To determine the effects of free bilirubin associated with transient hypoxia on developing rat neurons, the cells were exposed to bilirubin (0.25 to 5 micromol/L) and/or to hypoxia for 3 or 6 hours (95% N2-5% CO2). Glutamate receptor antagonists were added to some cultures. Cell death characteristics, energy metabolism, and protein synthesis were analyzed for 96 hours. Bilirubin increased apoptotic cell death. When associated with hypoxia, the neuronal loss was worsened. Bilirubin reduced energy metabolism, whereas a 6-hour exposure to hypoxia increased it for at least 24 hours, with no influence of additional bilirubin. Bilirubin with or without hypoxia induced 2 increases in protein synthesis, at 1 and 72 hours. In this model, bilirubin may promote programmed neuronal death. When bilirubin is associated with hypoxia, the deleterious effects are enhanced. The suppression of bilirubin induced neuronal damage by the NMDA (N-methyl-D-aspartate) receptor antagonist MK801 suggests the involvement of glutamate.