Takushi Hanita

Maternal Feeding Controls Fetal Biological Clock

Background It is widely accepted that circadian physiological rhythms of the fetus are affected by oscillators in the maternal brain that are coupled to the environmental light-dark (LD) cycle. Methodology/Principal Findings To study the link between fetal and maternal biological clocks, we investigated the effects of cycles of maternal food availability on the rhythms of Per1 gene expression in the fetal suprachiasmatic nucleus (SCN) and liver using a transgenic rat model whose tissues express luciferase in vitro. Although the maternal SCN remained phase-locked to the LD cycle, maternal restricted feeding phase-advanced the fetal SCN and liver by 5 and 7 hours respectively within the 22-day pregnancy. Conclusions/Significance Our results demonstrate that maternal feeding entrains the fetal SCN and liver independently of both the maternal SCN and the LD cycle. This indicates that maternal-feeding signals can be more influential for the fetal SCN and particular organ oscillators than hormonal signals controlled by the maternal SCN, suggesting the importance of a regular maternal feeding schedule for appropriate fetal molecular clockwork during pregnancy.

Novel modification of an artificial placenta: pumpless arteriovenous extracorporeal life support in a premature lamb model

Background:Previous studies aimed at developing an artificial placenta have had limited success. We hypothesized that the introduction of a high-performance membranous oxygenator to a pumpless artificial placenta could prolong the survival time of premature lambs.Methods:Immediately after delivery of the fetuses, the umbilical vessels were cannulated and connected to the pumpless artificial placenta. Both the fetuses and the circuit were submerged in a warm saline bath.Results:Five fetuses survived for 18.2 ± 3.2 (mean ± SEM) h after attachment to the artificial placenta, which maintained fetal circulation. Circuit blood flow was positively correlated with mean arterial pressure and negatively correlated with blood lactate levels. Milrinone administration transiently decreased lactate levels, although dopamine administration unexpectedly induced a marked increase in the lactate levels despite an elevated arterial pressure and improved circuit blood flow.Conclusion:We prolonged the survival of fetal lambs using a high-performance membranous oxygenator with a small priming volume. The increased systemic resistance induced by vasoconstrictors may increase the circuit blood flow excessively, resulting in circulation failure in systemic organs; therefore, vasodilators may be more useful than vasoconstrictors for maintaining organ blood flow within this circuit.

Effect of intrauterine inflammation on fetal cerebral hemodynamics and white-matter injury in chronically instrumented fetal sheep.

OBJECTIVE The purpose of this study was to analyze the effects of intrauterine inflammation on cerebral hemodynamics and white-matter injury in premature fetal sheep. STUDY DESIGN Fetuses were given an intravenous infusion of granulocyte colony-stimulating factor and an intraamniotic infusion of endotoxin; the fetuses were then assigned randomly to an acute hemorrhage group, an exchange transfusion group, or a control group. During each insult, the cerebral hemodynamics were assessed with near-infrared spectroscopy. Finally, the fetuses were processed for neuropathologic analysis and compared statistically. RESULTS Necrotizing funisitis and chorioamnionitis were induced in all the fetuses. A significant decrease in the blood oxygen content and an increase in the brain total hemoglobin level were observed after the endotoxin infusion. Soon after hemodynamic insult, the fetuses in both the acute hemorrhage and the exchange transfusion groups showed an abrupt decrease in the total brain hemoglobin level; 4 of the 5 fetuses in each treatment group, but none of the fetuses in the control group, exhibited periventricular leukomalacia. CONCLUSION Hemorrhagic hypotension or anemic hypoxemia might induce a sudden cessation of fetal brain-sparing effects through progressive inflammatory hypoxemia, which results in focal white-matter injuries.

Designing Artificial Environments for Preterm Infants Based on Circadian Studies on Pregnant Uterus

Using uterine explants from Per1::Luc rats and in situ hybridization, we recently reported that the circadian property of the molecular clock in the uterus and placenta is stably maintained from non-pregnancy, right through to the end stage of pregnancy under regular light-dark (LD) cycles. Despite long-lasting increases in progesterone during gestation and an increase in estrogen before delivery, the uterus keeps a stable Per1::Luc rhythm throughout the pregnancy. The study suggests the importance of stable circadian environments for fetuses to achieve sound physiology and intrauterine development. This idea is also supported by epidemiological and animal studies, in which pregnant females exposed to repeated shifting of the LD cycles have increased rates of reproductive abnormalities and adverse pregnancy outcomes. Leading from this, we introduced artificial circadian environments with controlled lighting conditions to human preterm infants by developing and utilizing a specific light filter which takes advantage of the unique characteristics of infants’ developing visual photoreceptors. In spite of growing evidence of the physiological benefits of nighttime exposure to darkness for infant development, many Japanese Neonatal Intensive Care Units (NICUs) still prefer to maintain constant light in preparation for any possible emergencies concerning infants in incubators. To protect infants from the negative effects of constant light on their development in the NICU, we have developed a new device similar to a magic mirror, by which preterm infants can be shielded from exposure to their visible wavelengths of light even in the constant light conditions of the NICU while simultaneously allowing medical care staff to visually monitor preterm infants adequately. The device leads to significantly increased infant activity during daytime than during night time and better weight gains.

Mutation analysis of SOX9 and single copy number variant analysis of the upstream region in eight patients with campomelic dysplasia and acampomelic campomelic dysplasia

Mutation Analysis of SOX9 and Single Copy Number Variant Analysis of the Upstream Region in Eight Patients With Campomelic Dysplasia and Acampomelic Campomelic Dysplasia Yuka Wada,* Gen Nishimura, Toshiro Nagai, Hideaki Sawai, Mayumi Yoshikata, Shinichirou Miyagawa, Takushi Hanita, Seiji Sato, Tomonobu Hasegawa, Shumpei Ishikawa, and Tsutomu Ogata Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, Tokyo, Japan Department of Radiology, Tokyo Metropolitan Kiyose Children’s Hospital, Kiyose, Japan Department of Pediatrics, Dokkyo Medical University, Koshigaya, Japan Genetic Counseling and Clinical Research Unit, Kyoto University, Kyoto, Japan Department of Neonatology, Hyogo Children’s Hospital, Kobe, Japan Department of Pediatrics, National Hospital Organization Kure Medical Center, Kure, Japan Department of Pediatrics, Tohoku University School of Medicine, Sendai, Japan Department of Pediatrics, Saitama City Hospital, Saitama, Japan Department of Pediatrics, School of Medicine, Keio University, Tokyo, Japan Genome Science Division, Department of Pathology, Research Center for Advanced Science and Technology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Daily ethanol exposure during late ovine pregnancy: physiological effects in the mother and fetus in the apparent absence of overt fetal cerebral dysmorphology

High levels of ethanol (EtOH) consumption during pregnancy adversely affect fetal development; however, the effects of lower levels of exposure are less clear. Our objectives were to assess the effects of daily EtOH exposure (3.8 USA standard drinks) on fetal-maternal physiological variables and the fetal brain, particularly white matter. Pregnant ewes received daily intravenous infusions of EtOH (0.75 g/kg maternal body wt over 1 h, 8 fetuses) or saline (8 fetuses) from 95 to 133 days of gestational age (DGA; term ∼145 DGA). Maternal and fetal arterial blood was sampled at 131-133 DGA. At necropsy (134 DGA) fetal brains were collected for analysis. Maternal and fetal plasma EtOH concentrations reached similar maximal concentration (∼0.11 g/dl) and declined at the same rate. EtOH infusions produced mild reductions in fetal arterial oxygenation but there were no changes in maternal oxygenation, maternal and fetal Pa(CO(2)), or in fetal mean arterial pressure or heart rate. Following EtOH infusions, plasma lactate levels were elevated in ewes and fetuses, but arterial pH fell only in ewes. Fetal body and brain weights were similar between groups. In three of eight EtOH-exposed fetuses there were small subarachnoid hemorrhages in the cerebrum and cerebellum associated with focal cortical neuronal death and gliosis. Overall, there was no evidence of cystic lesions, inflammation, increased apoptosis, or white matter injury. We conclude that daily EtOH exposure during the third trimester-equivalent of ovine pregnancy has modest physiological effects on the fetus and no gross effects on fetal white matter development.

A Parallelized Pumpless Artificial Placenta System Significantly Prolonged Survival Time in a Preterm Lamb Model

An artificial placenta (AP) is an arterio-venous extracorporeal life support system that is connected to the fetal circulation via the umbilical vasculature. Previously, we published an article describing a pumpless AP system with a small priming volume. We subsequently developed a parallelized system, hypothesizing that the reduced circuit resistance conveyed by this modification would enable healthy fetal survival time to be prolonged. We conducted experiments using a premature lamb model to test this hypothesis. As a result, the fetal survival period was significantly prolonged (60.4 ± 3.8 vs. 18.2 ± 3.2 h, P < 0.01), and circuit resistance and minimal blood lactate levels were significantly lower in the parallel circuit group, compared with our previous single circuit group. Fetal physiological parameters remained stable until the conclusion of the experiments. In summary, parallelization of the AP system was associated with reduced circuit resistance and lactate levels and allowed preterm lamb fetuses to survive for a significantly longer period when compared with previous studies.

Alcohol exposure during late gestation: Multiple developmental outcomes in sheep

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life.

Induction of Necrotizing Funisitis by Fetal Administration of Intravenous Granulocyte-Colony Stimulating Factor and Intra-Amniotic Endotoxin in Premature Fetal Sheep

The purpose of the present study was to determine whether experimental intrauterine inflammation could induce necrotizing funisitis, a severe, chronic inflammation of the umbilical cord. Fetuses, randomly divided into four groups (n = 4 each), were infused with 50 μg/d of granulocyte-colony stimulating factor (G-CSF) intravenously on d 125–129 of gestation (G-CSF group), 20 mg of endotoxin into the amniotic cavity on d 127 gestation (endotoxin group), both G-CSF and endotoxin (G-CSF + endotoxin group), or only saline (control group). On d 130 of gestation, the umbilical cords were processed for histologic analysis, scored for degree of inflammation, and compared statistically. At birth, the blood polymorphonuclear leukocyte counts in G-CSF and G-CSF + endotoxin groups were significantly higher than those in endotoxin and control groups (p < 0.05). The inflammatory score of the umbilical cord in G-CSF + endotoxin group was significantly higher than those in the other three groups (p < 0.05). All the fetuses in G-CSF + endotoxin group had necrotizing funisitis, but none of the fetuses in the other three groups developed this condition. An increase in blood polymorphonuclear leukocytes before their activation in the umbilical cord is probably essential for experimentally inducing necrotizing funisitis.

Long-Term Pulmonary Effects of Intrauterine Exposure to Endotoxin Following Preterm Birth in Sheep

Our aim was to determine whether fetal exposure to intraamniotic lipopolysaccharide (LPS) persistently alters the lungs following moderate preterm birth. Fetal sheep were exposed to LPS (1 mg/d) or saline from 0.75 to preterm birth at 0.90 of gestation. Eleven weeks after preterm birth, lung structure was unaltered. Interleukin (IL)-1β messenger RNA (mRNA) levels were elevated in lungs of LPS-exposed lambs (P < .05) but IL-1β protein levels were unaltered. Lung mRNA levels of IL-6, IL-8 and tumor necrosis factor α, and percentage of inflammatory cells were not different between groups. Surfactant protein (SP)-A and SP-C mRNA levels and SP-B tissue protein expression were higher in LPS-exposed lambs than controls (all P < .05); however, expression of SP-A and SP-C proteins was reduced. Prenatal LPS exposure causes a persistent increase in gene expression of proinflammatory mediators and surfactant proteins and a decrease in lung tissue SP-A and -C protein expression after preterm birth, which may affect lung immunity.