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 (H 2 ) administration. We have previously demonstrated in the MIA mouse model that maternal administration of H 2 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, threechamber, 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 H 2 administration markedly attenuated these LPS-induced abnormalities. Moreover, we evaluated the effect of H 2 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 H 2 -administered dams. In primary cultured astrocytes, LPS-induced pro-inflammatory cytokines were attenuated by H 2 administration. Overall, these findings indicate that maternal H 2 administration exerts neuroprotective effects and ameliorates MIA-induced neurodevelopmental deficits of offspring later in life. Published: xx xx xxxx OPEN www.nature.com/scientificreports/ 2 ScIENtIfIc REPORTS | (2018) 8:9221 | lead to direct and indirect death or dysfunction of neuronal and oligodendrocyte cells 10 , which are considered to be the main targets of fetal brain injury 11 . We have previously suggested that the maternal administration of molecular hydrogen (H 2 ) plays a neuroprotective role in the fetal brain against injury caused by oxidative stress and inflammation 12-14 . Ohsawa et al. discovered that H 2 acts as an antioxidant that selectively neutralizes hydroxyl radicals (•OH) and protects the brain from injury 15 . H 2 reacts with strong oxidants, such as •OH, but remains mild enough to neither disturb the metabolic redox reactions nor affect ROS signaling. Several subsequent studies have explored the therapeutic and preventive effects of H 2 , and have indicated that H 2 has other properties as well, including anti-inflammatory and anti-apoptotic effects 16, 17 . The safety and some promising benefits of H 2 have already been demonstrated, not only in animal models but also in patients, including those with Parkinson's disease 18 , diabetes mellitus 19 and mild cognitive impairment 20 , although no large randomized controlled trials have been conducted to date. Recently, we demonstrated that the administration of H 2 to pregnant mouse dams significantly increased H 2 concentration in the fetal brain, through the maternal-fetal interface, in a mouse model of lipopolysaccharide (LPS)-induced MIA 12 . Fetal damage in this MIA model and the effect of maternal H 2 administration on this damage can be summarized as follows 12 : (1) high mortality rate of MIA fetuses, (2) brain injury associated with elevated levels of pro-inflammatory cytokines, aberrant microglial activation, and oxidative damage in the MIA fetal brains, and (3) attenuation of these adverse outcomes by maternal H 2 administration. Thus, the protective effect of maternal H 2 administration on short-term outcomes in the MIA offspring has been demonstrated. However, long-term outcomes of the surviving offspring are yet to be investigated. Therefore, the present study investigated long-term neurological outcomes in the MIA mouse model and the efficacy of H 2 administration in pregnant dams, using both histological and behavioral examinations. Astrocytes, which are the most abundant cell population in the CNS, play a key role in normal brain development 21, 22 . However, when astrocytes are exposed to maternally-derived cytokines in MIA models, they secrete various cytokines that lead to neurodevelopmental impairments 23 . Moreover, activation of astrocytes is known to be a factor in the prolongation of brain damage, and contributes to the formation of glial scars that limit neuronal plasticity 24 . Thus, in the present study, the MIA-related astrocytic activation and its modification by H 2 administration were also investigated, both in vivo and in vitro. Maternal H 2 administration restored offspring growth. We investigated postnatal growth, which could be related to neurologic outcome. From postnatal day (P) 6 to P10, offspring growth was significantly retarded in the LPS group than in the Control group (p < 0.05). Although significant differences were not observed between the LPS and LPS + HW group, maternal administration of Hydrogen water (HW) restored offspring growth to the control level (Fig. 1) .
Output: Administration of molecular hydrogen during pregnancy improves behavioral abnormalities of offspring in a maternal immune activation model OPEN