Takafumi Ushida

Maternal molecular hydrogen treatment attenuates lipopolysaccharide-induced rat fetal lung injury

Abstract Maternal inflammation is associated with spontaneous preterm birth and respiratory impairment among premature infants. Recently, molecular hydrogen (H2) has been reported to have a suppressive effect on oxidative stress and inflammation. The aim of this study was to evaluate the effects of H2 on fetal lung injury caused by maternal inflammation. Cell viability and the production of interleukin-6 (IL-6) and reactive oxygen species (ROS) were examined by treatment with lipopolysaccharide (LPS) contained in ordinal or H2-rich medium (HM) using a human lung epithelial cell line, A549. Pregnant Sprague Dawley rats were divided into three groups: Control, LPS, and HW + LPS groups. Rats were injected with phosphate-buffered saline (Control) or LPS intraperitoneally (LPS) on gestational day 19 and provided H2 water (HW) ad libitum for 24 h before LPS injection (HW + LPS). Fetal lung samples were collected on day 20, and the levels of apoptosis, oxidative damage, IL-6, and vascular endothelial growth factor (VEGF) were evaluated using immunohistochemistry. The number of apoptotic cells, and levels of ROS and IL-6 were significantly increased by LPS treatment, and repressed following cultured with HM in A549 cells. In the rat models, the population positive for cleaved caspase-3, 8-hydroxy-2′-deoxyguanosine, IL-6, and VEGF was significantly increased in the LPS group compared with that observed in the Control group and significantly decreased in the HW + LPS group. In this study, LPS administration induced apoptosis and oxidative damage in fetal lung cells that was ameliorated by maternal H2 intake. Antenatal H2 administration may decrease the pulmonary mobility associated with inflammation in premature infants.

Neuroprotective potential of molecular hydrogen against perinatal brain injury via suppression of activated microglia.

Exposure to inflammation in utero is related to perinatal brain injury, which is itself associated with high rates of long-term morbidity and mortality in children. Novel therapeutic interventions during the perinatal period are required to prevent inflammation, but its pathogenesis is incompletely understood. Activated microglia are known to play a central role in brain injury by producing a variety of pro-inflammatory cytokines and releasing oxidative products. The study is aimed to investigate the preventative potential of molecular hydrogen (H2), which is an antioxidant and anti-inflammatory agent without mutagenicity. Pregnant ICR mice were injected with lipopolysaccharide (LPS) intraperitoneally on embryonic day 17 to create a model of perinatal brain injury caused by prenatal inflammation. In this model, the effect of maternal administration of hydrogen water (HW) on pups was also evaluated. The levels of pro-inflammatory cytokines, oxidative damage and activation of microglia were determined in the fetal brains. H2 reduced the LPS-induced expression of pro-inflammatory cytokines, oxidative damage and microglial activation in the fetal brains. Next, we investigated how H2 contributes to neuroprotection, focusing on microglia, using primary cultured microglia and neurons. H2 prevented LPS- or cytokine-induced generation of reactive oxidative species by microglia and reduced LPS-induced microglial neurotoxicity. Finally, we identified several molecules influenced by H2, involved in the process of activating microglia. These results suggested that H2 holds promise for the prevention of inflammation related to perinatal brain injury.

Maternal molecular hydrogen administration on lipopolysaccharide-induced mouse fetal brain injury

Fetal brain injury is often related to prenatal inflammation; however, there is a lack of effective therapy. Recently, molecular hydrogen (H2), a specific antioxidant to hydroxyl radical and peroxynitrite, has been reported to have anti-inflammatory properties. The aim of this study was to investigate whether maternal H2 administration could protect the fetal brain against inflammation. Pregnant C3H/HeN mice received an intraperitoneal injection of lipopolysaccharide (LPS) on gestational day 15.5 and were provided with H2 water for 24 h prior to LPS injection. Pup brain samples were collected on gestational day 16.5, and the levels of apoptosis and oxidative damage were evaluated using immunohistochemistry. Interleukin-6 (IL-6) levels were examined using real-time PCR. The levels of apoptosis and oxidative damage, as well as the levels of IL-6 mRNA, increased significantly when the mother was injected with LPS than that in the control group. However, these levels were significantly reduced when H2 was administered prior to the LPS-injection. Our results suggest that LPS-induced apoptosis, oxidative damage and inflammation in the fetal brain were ameliorated by maternal H2 administration. Antenatal H2 administration might protect the premature brain against maternal inflammation.

Molecular hydrogen ameliorates several characteristics of preeclampsia in the Reduced Uterine Perfusion Pressure (RUPP) rat model

Oxidative stress plays an important role in the pathogenesis of preeclampsia. Recently, molecular hydrogen (H2) has been shown to have therapeutic potential in various oxidative stress-related diseases. The aim of this study is to investigate the effect of H2 on preeclampsia. We used the reduced utero-placental perfusion pressure (RUPP) rat model, which has been widely used as a model of preeclampsia. H2 water (HW) was administered orally ad libitum in RUPP rats from gestational day (GD) 12-19, starting 2 days before RUPP procedure. On GD19, mean arterial pressure (MAP) was measured, and samples were collected. Maternal administration of HW significantly decreased MAP, and increased fetal and placental weight in RUPP rats. The increased levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and diacron reactive oxygen metabolites as a biomarker of reactive oxygen species in maternal blood were decreased by HW administration. However, vascular endothelial growth factor level in maternal blood was increased by HW administration. Proteinuria, and histological findings in kidney were improved by HW administration. In addition, the effects of H2 on placental villi were examined by using a trophoblast cell line (BeWo) and villous explants from the placental tissue of women with or without preeclampsia. H2 significantly attenuated hydrogen peroxide-induced sFlt-1 expression, but could not reduce the expression induced by hypoxia in BeWo cells. H2 significantly attenuated sFlt-1 expression in villous explants from women with preeclampsia, but not affected them from normotensive pregnancy. The prophylactic administration of H2 attenuated placental ischemia-induced hypertension, angiogenic imbalance, and oxidative stress. These results support the theory that H2 has a potential benefit in the prevention of preeclampsia.

The expression of Toll-like receptor 5 in preterm histologic chorioamnionitis

Spontaneous preterm birth is often caused by chorioamnionitis. Toll-like receptors (TLRs) have a role in the response of the innate immune system. The role of TLR5 in chorioamnionitis remains unclear: however, TLR5 was reported to have a significantly stronger effect on the induction of interleukin (IL)-6 when compared with other TLRs in amniotic epithelial cells. The aim of this study was to investigate TLR5 expression in placentas with preterm histologic chorioamnionitis (HCA). The expression levels of TLR5 were evaluated in the amnions, chorions, deciduae and villi with and without HCA using immunohistochemistry. The co-localization of IL-6 or IL-8 with TLR5 was examined by immunofluorescence. The production of IL-6 was examined in primary tissue cultured fetal membranes treated with and without the TLR5 agonist. The protein expression of TLR5 was significantly increased in amnions with HCA (p<0.05) and showed a trend toward an increase in chorions with HCA, whereas no significant difference was detected in the villi and decidua. TLR5 co-localized with IL-6 and IL-8 in amnions and chorions. IL-6 showed a significant increase (p<0.05) with the TLR5 agonist. These results suggest that TLR5 plays a role in the pathogenesis of preterm HCA and IL-6 production.

Administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model

The aim of the present study was to investigate long-term outcomes of the offspring in a lipopolysaccharide (LPS)-induced maternal immune activation (MIA) model and the effect of maternal molecular hydrogen (H2) administration. We have previously demonstrated in the MIA mouse model that maternal administration of H2 attenuates oxidative damage and neuroinflammation, including induced pro-inflammatory cytokines and microglial activation, in the fetal brain. Short-term memory, sociability and social novelty, and sensorimotor gating were evaluated using the Y-maze, three-chamber, and prepulse inhibition (PPI) tests, respectively, at postnatal 3 or 4 weeks. The number of neurons and oligodendrocytes was also analyzed at postnatal 5 weeks by immunohistochemical analysis. Offspring of the LPS-exposed dams showed deficits in short-term memory and social interaction, following neuronal and oligodendrocytic loss in the amygdala and cortex. Maternal H2 administration markedly attenuated these LPS-induced abnormalities. Moreover, we evaluated the effect of H2 on LPS-induced astrocytic activation, both in vivo and in vitro. The number of activated astrocytes with hypertrophic morphology was increased in LPS-exposed offspring, but decreased in the offspring of H2-administered dams. In primary cultured astrocytes, LPS-induced pro-inflammatory cytokines were attenuated by H2 administration. Overall, these findings indicate that maternal H2 administration exerts neuroprotective effects and ameliorates MIA-induced neurodevelopmental deficits of offspring later in life.

Maternal protein S activity is associated with massive hemorrhage during caesarean section: retrospective case-control study

Abstract Objective: The objective of this study is to identify any correlation between the maternal protein S (PS) activity, which decreases spontaneously during normal pregnancy, and the pregnancy outcome, especially the amount of bleeding during caesarean section (CS). Materials and methods: We analyzed 129 pregnant women who received elective CS at our hospital which is a tertiary perinatal center. The relationship between the amount of intraoperative hemorrhage and PS activity was estimated by simple linear regression. Univariate and multivariate analyses were also performed to determine whether or not the maternal PS activity was associated with massive hemorrhage (>1000 g). Results: The maternal PS activity correlated with the amount of intraoperative hemorrhage (p = .048, r = 0.175) and could be an independent predictor of massive hemorrhage (odds ratio 1.06; 95% confidence interval 1.01–1.12; p = .013). Conclusions: Maternal PS activity was associated with the amount of hemorrhage during CS and the occurrence of massive hemorrhage. PS activity reduction naturally occurring during pregnancy could contribute to alleviation of massive hemorrhage.

The rate of neonatal respiratory distress syndrome/transient tachypnea in the newborn and the amniotic lamellar body count in twin pregnancies compared with singleton pregnancies

BACKGROUND Whether or not the period of fetal lung maturity differs between twin and singleton pregnancies has not been clarified. We examined whether or not fetal lung maturity and fetal lung absorption are achieved earlier in twin fetuses than in singleton fetuses. METHODS We registered 454 singleton pregnancies and 398 twin pregnancies with no congenital abnormalities affecting the respiratory function or neonatal deaths. All patients were delivered by Caesarean section without labor between 24 and 38 gestational weeks. The amniotic fluid samples were analyzed immediately without centrifugation. A multiple logistic regression analysis was performed to explore the relationship between twin pregnancy and neonatal respiratory distress syndrome and transient tachypnea of the newborn (RDS/TTN). RESULTS The rate of RDS/TTN in infants was significantly higher and the lamellar body counts (LBCs) significantly lower in singleton pregnancies than that in twin pregnancies (P < .001). According to a multivariate logistic regression analysis, twin pregnancy (odds ratio, 0.34; 95% confidence interval, 0.22-0.55) was a significant preventive factor for neonatal RDS/TTN. CONCLUSIONS We showed that twin fetuses experience more rapid lung maturation and lung fluid absorption than singleton fetuses, as confirmed by the higher LBC values in twin fetuses.

The impact of fertility treatment on the neonatal respiratory outcomes and amniotic lamellar body counts in twin pregnancies

BACKGROUND To elucidate the impact of fertility treatment on neonatal respiratory outcomes and amniotic lamellar body counts (LBCs) in twin pregnancies. METHODS One hundred ninety twin pairs, including 99 dichorionic twin (DCT) and 91 monochorionic twin (MCT) pairs were registered at our institutions. All amniotic fluid samples were obtained from each sac at cesarean section. Samples were analyzed immediately after arrival at the laboratory without centrifugation. We divided the patients into 3 groups: the no therapy group (natural conception), the induced ovulation group (with or without intrauterine insemination), and the assisted reproductive technology (ART) group (in vitro fertilization or intracytoplasmic sperm injection). RESULTS No statistically significant associations between the fertility treatment and the rates of neonatal RDS/TTN were observed in the whole study population (odds ratio [OR], 0.95; 95% confidence interval [CI], 0.45-2.00), DCT (OR, 0.86; 95%CI, 0.30-2.47), and MCT (OR, 1.45; 95%CI, 0.41-5.11). In addition, there was no association between the fertility treatment and neonatal RDS/TTN in the propensity score analysis of the whole study population (OR, 1.25; 95%CI, 0.57-2.74). CONCLUSIONS None of the individual types of fertility treatment had a direct impact on respiratory disorders such as RDS and TTN in twin infants.

Effect of molecular hydrogen on uterine inflammation during preterm labour

Intrauterine inflammation causes preterm birth and is associated with complications in preterm neonates. Thus, strategies aimed at suppressing inflammation are expected to be effective for reducing the risk of preterm birth and associated complications. Our previous studies demonstrated that molecular hydrogen (H2), an anti-inflammatory agent, prevented inflammation-induced impairment in foetal brain and lung tissues in lipopolysaccharide (LPS)-induced rodent models. However, it remains unclear whether H2 is capable of inhibiting preterm labour. The aim of the current study was therefore to investigate the effect of H2 on inflammation-induced preterm labour. Pregnant ICR (CD-1) mice were divided into three groups: Control, LPS and H2 water (HW) + LPS. In the control and LPS groups, vehicle and LPS, respectively, were intraperitoneally injected on embryonic day 15.5. In the HW + LPS group, HW was administered 24 h prior to LPS injection. The time from LPS administration to parturition was compared between the LPS and HW + LPS groups. Maternal uterus was collected 6 h after LPS injection and the transcript levels of pro-inflammatory cytokines, contractile-associated proteins (CAPs), matrix metalloproteinase-3 (Mmp3) and endothelin-1 (Et1) were assessed by reverse transcription-quantitative polymerase chain reaction. The protein levels of cyclooxygenase-2 (Cox2) were also evaluated by immunohistochemistry. The time from LPS administration to parturition in the HW + LPS group was significantly increased compared with that in the LPS group (33.5±3.4 vs. 18.3±8.8 h, respectively, P=0.020). H2 administration also resulted in significantly higher progesterone levels compared with LPS treatment alone (P=0.002). The transcript levels of pro-inflammatory cytokines, CAPs, Mmp3 and Et1 in the uteri of the LPS group were significantly higher than those in the control group (all P<0.05). In turn, all these levels with the exception of interleukin-8 and Mmp3 were significantly lower in the HW + LPS group compared with those in the LPS group (all P<0.05). The protein levels of Cox2 in the LPS group were also significantly increased compared with those in the control (P<0.001) and HW + LPS (P=0.003) groups. These results suggest that inflammation-induced changes in the uterus may be ameliorated through maternal H2 administration. Preventive H2 administration may therefore represent an effective strategy for the suppression of inflammation during preterm labour.