Neonatal Stage Research Articles

Prevention of Cerebral Palsy with Novel Hypoxia Index Composed of Fetal Heart Rate Deceleration

Fetal brain damage develops after the loss of FHR variability followed by infantile cerebral palsy due to severe hypoxia in frequently repeated fetal heart rate (FHR) decelerations (transient bradycardia) or prolonged fetal bradycardia, where novel hypxia index is 25 or more, and it is prevented if the hpoxia index is 24 or less. The hypoxia index (HI) is the sum of FHR deceleration durations (min) divided by the lowest FHR (bpm), and multiplied by 100 (Figure 1). The HI is calculated by visual measurement, while it is also suitably calculated by computerized FHR monitoring. Cerebral palsy is prevented when HI is 24 or less with almost zero error probability in the delivery. The cases whose HI was 25 or more will develop cerebral palsy, thus, it can receive early cerebral palsy trearments in neonatal stage. As late deceleration disappeared when the parturient woman changed her posture to lateral one from supine, a parturient woman is recommended to have lateral posture, when they notice the appearance of FHR deceleration during the delivery to disappear deceleration to prevent the increase of HI value. As the HI is adopted not only late deceleration, but also all decelerations and continuous bradycardia, fetal diagnosis will change to objective numeric FHR analysis from the monitoring with vague subjective FHR pattern classification.

Changes in serum pepsinogen levels and their value as a predictor of treatment outcomes in children with peptic ulcer.

To investigate pepsinogen secretion from the neonatal stage to childhood and its diagnostic value for peptic ulcer (PU) in children. In this study, 2114 'healthy' children and 51 children with PUs undergoing a medical examination by gastroscopy were selected as subjects. The serum content of pepsinogen I (PGI) and serum pepsinogen II (PGII) was tested for each of the subjects using time-resolved fluorescence immunoassay, which is characterised by high sensitivity and a wide measuring range. The serum PGI and PGII levels were found to increase with age, becoming stable and similar to those of adults at the age of 16. In 51 children with PUs, PGI was 201.03 ± 30.74 ng/mL before treatment and 187.92 ± 19.86 ng/mL after treatment (P > 0.05); PGII was 17.36 ± 1.47 ng/mL before treatment and 17.20 ± 3.98 ng/mL after treatment (P > 0.05). It is difficult to establish the normal range of PG in children owing to its variance by age. However, if the normal reference range for individual age groups is known, it may still serve as a useful diagnosis system as well as a detecting indicator during the course of PU treatment. There are significant differences in PGI expression in children with PU before and after PU is cured, whereas other indicators show no differences before and after treatment.

Comparison of birth weight and umbilical and placental characteristics of cloned and artificial insemination-derived piglets

Somatic cell nuclear transfer (SCNT)-derived piglets have significantly higher stillbirth rate and postnatal mortality rate than artificial insemination (AI)-generated piglets. The question whether the low survival rate of SCNT piglets was related to birth weight, umbilical cord or placenta development was investigated. In this study, stillbirth rate, neonatal death rate, birth weight, umbilical cord status, placental parameters and placental gene expression patterns were compared between SCNT and AI piglets. Results showed that mortality rates at birth and during the neonatal stage of SCNT piglets were significantly higher than those of AI piglets. The incidence of abnormal umbilical cord in SCNT and SCNT-liveborn (SCNT-LB) piglets was significantly higher than in AI and AI-liveborn (AI-LB) piglets. Birth weight, placental weight, placental surface area and placental efficiency in SCNT and SCNT-LB piglets were significantly lower than those of AI and AI-LB piglets. Placental expression profiles of imprinting, angiopoiesis and nutrient transport-related genes were defective in SCNT-LB piglets compared with those in AI-LB piglets. Thus, the low survival rate of SCNT piglets may be associated with abnormal umbilical cord and placenta development. These characteristics may have resulted from aberrant expression of angiogenesis, nutrient transport, and imprinting-related genes in the placentas.

Suppressor of Fused regulates the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism.

ABSTRACTThe ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (Sufu) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B1 cells. In contrast, we found a significant decrease in Gsx2+ and a more dramatic decrease in Tbr2+ transit amplifying cells (TACs) indicating that innate differences between dorsal and ventral forebrain derived Type B1 cells influence Sufu function. However, many precursors accumulated in the dorsal V-SVZ or failed to survive, demonstrating that despite the over-proliferation of Type B1 cells, they are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression and surprisingly, a significant downregulation of Sonic hedgehog (Shh) signaling. Therefore, these findings indicate a potential role of the Sufu-Gli3 regulatory axis in the neonatal dorsal V-SVZ independent of Shh signaling in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ.

Transcriptional response to Wnt activation regulates the regenerative capacity of the mammalian cochlea.

ABSTRACTLack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.

Maternal vitamin D3 status & association with low birth weight infant

Background and Objective: Maternal vitamin D levels may affect fetal growth and cause adverse pregnancy outcomes including low birth weight in neonatal stage. This study was designed to evaluate and determine if the deficiency of maternal Vitamin D associated and affected the growth development of birth weight of neonates. Methods: This study was carried-out on 200 neonates through a cross-sectional study, that used data from the nursery ward at maternity teaching hospital hospitals in Kurdistan, Iraq during a one year period from March 2016 to March 2017. The neonates were divided into two group two groups, neonates with birth weight 2500 gr (n = 55). We depend on the data classifications on medical history, physical examination using a questionnaire method for neonate classifications. Birth time blood samples of their mothers were analyzed for serum 25-(OH)-vitamin D using enzyme linked immunosorbent assay method. Maternal vitamin D status was compared in the two groups. Results: The mean maternal vitamin D level was 24 nmol/L. for mothers with low birth weight and 22 (48.89 %) for mothers with vitamin D deficiency. Seventeen (37.77 %) and 6 (13.34 %) of the mothers with low birth weight had insufficient vitamin D and 6 (13.34 %) had normal Vitamin D levels. The mothers with normal birth weight had a mean vitamin D level of 38.10 nmol/L and the mothers of low levels reached to 10 nmol/L (18.18 %), those of insufficiency vitamin D was 20 nmol/L (36.36 %) and those of sufficient vitamin D 25 nmol/L (45.46 %). Conclusion: Mothers with low vitamin D levels are more likely to have neonates with low birth weight. Increasing the vitamin D levels through nutrition is important for improving the neonatal body weight.

SMAD4 Deficiency Leads to Development of Arteriovenous Malformations in Neonatal and Adult Mice.

BackgroundHereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disorder caused by mutations in endoglin (ENG), activin receptor‐like kinase 1 (ACVRL1;ALK1), or SMAD4. Major clinical symptoms of HHT are arteriovenous malformations (AVMs) found in the brain, lungs, visceral organs, and mucosal surface. Animal models harboring mutations in Eng or Alk1 recapitulate all of these HHT clinical symptoms and have been useful resources for studying mechanisms and testing potential drugs. However, animal models representing SMAD4 mutations have been lacking. The goal of this study is to evaluate Smad4‐inducible knockout (iKO) mice as an animal model of HHT and compare the phenotypes with other established HHT animal models.Methods and ResultsGlobal Smad4 deletion was induced at neonatal and adult stages, and hemoglobin levels, gastrointestinal hemorrhage, and presence of aberrant arteriovenous connections were examined. Neonatal Smad4‐iKO mice exhibited signs of gastrointestinal bleeding and AVMs in the brain, intestine, nose, and retina. The radial expansion was decreased, and AVMs were detected on both distal and proximal retinal vasculature of Smad4‐iKOs. Aberrant smooth muscle actin staining was observed in the initial stage AVMs and their connecting veins, indicating abnormal arterial flow to veins. In adult mice, Smad4 deficiency caused gastrointestinal bleeding and AVMs along the gastrointestinal tract and wounded skin. HHT‐related phenotypes of Smad4‐iKOs appeared to be comparable with those found in Alk1‐iKO and Eng‐iKO mice.ConclusionsThese data further confirm that SMAD signaling is crucial for normal arteriovenous network formation, and Smad4‐iKO will be an alternative animal model of AVM research associated with HHT.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice.

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

Role of androgens in energy metabolism affecting on body composition, metabolic syndrome, type 2 diabetes, cardiovascular disease, and longevity: lessons from a meta-analysis and rodent studies.

Testosterone is a sex hormone produced by testicular Leydig cells in males. Blood testosterone concentrations increase at three time-periods in male life-fetal, neonatal (which can be separated into newborn and infant periods), and pubertal stages. After peaking in the early 20s, the blood bioactive testosterone level declines by 1-2% each year. It is increasingly apparent that a low testosterone level impairs general physical and mental health in men. Here, this review summarizes recent systematic reviews and meta-analyses of epidemiological studies in males (including cross-sectional, longitudinal, and androgen deprivation studies, and randomized controlled testosterone replacement trials) in relation to testosterone and obesity, body composition, metabolic syndrome, type 2 diabetes, cardiovascular disease, and longevity. Furthermore, underlying mechanisms are discussed using data from rodent studies involving castration or androgen receptor knockout. This review provides an update understanding of the role of testosterone in energy metabolism. Abbreviations AR: androgen receptor; CV: cardiovascular; FDA: US Food and Drug Administration; HFD: high-fat diet; KO: knockout; MetS: metabolic syndrome; RCT: randomized controlled trial; SHBG: sex hormone binding globulin; SRMA: systematic review and meta-analysis; TRT: testosterone replacement therapy; T2DM:type 2 diabetes mellitus.

Janus kinase JAK1 maintains the ovarian reserve of primordial follicles in the mouse ovary

Is the Janus kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway involved in ovarian follicle development and primordial follicle activation? JAK1 is a key factor involved in the regulation of primordial follicle activation and maintenance of the ovarian reserve. A series of integrated, intrinsic signalling pathways (including PI3K/AKT, mTOR and KITL) are responsible for regulating the ovarian reserve of non-growing primordial follicles and ultimately female fertility. The JAK-STAT signal transduction pathway is highly conserved with established roles in cell division and differentiation. Key pathway members (specifically JAK1, STAT3 and SOCS4) have been previously implicated in early follicle development. A laboratory animal study was undertaken using the C57Bl/6 inbred mouse strain as a model for human ovarian follicle development. To determine which Jak genes were most abundantly expressed during primordial follicle activation, mRNA expression was analysed across a developmental time-course, with ovaries collected from female mice at post-natal days 1 (PND1), 4 (PND4), 8 (PND8), as well as at 6 weeks (6WK) and 7 months (7MTH) (n ≥ 4). Functional analysis of JAK1 was performed on PND2 mouse ovaries subjected to in vitro explant culture treated with 12.5 μM Ruxolitinib (JAK inhibitor) or vehicle control (DMSO) for 48 h prior to histological assessment (n ≥ 4). The expression and localization of the JAK family during ovarian follicle development in the C57Bl/6 inbred mouse strain were evaluated using quantitative PCR, immunoblotting and immunolocalisation. Functional studies were undertaken using the JAK inhibitor Ruxolitinib to investigate the underpinning cellular mechanisms via biochemical in vitro inhibition and histological assessment of intact neonate ovaries. All experiments were replicated at least three times using tissue from different mice unless otherwise stated. Jak1 is the predominant Jak mRNA expressed in the C57Bl/6 mouse ovary across all developmental time-points assessed (P ≤ 0.05). Forty-eight hour inhibition of JAK1 with Ruxolitinib of PND2 ovaries in vitro demonstrated concomitant acceleration of primordial follicle activation and apoptosis (P ≤ 0.001) and upregulation of downstream JAK-STAT pathway members STAT3 and suppressors of cytokine signalling 4 (SOCS4). N/A. Results are shown in one species, the C57Bl/6 mouse strain as an established model of human ovary development. Ruxolitinib also inhibits JAK2, with decreased efficacy. However, Jak2 mRNA had limited expression in the mouse ovary, particularly at the neonatal stages of follicle development, thus any effect of Ruxolitinib on primordial follicle activation was unlikely to be mediated via this isoform. This study supports a key role for JAK1 in the maintenance and activation of primordial follicles, with potential for targeting the JAK-STAT pathway as a method of regulating the ovarian reserve and female fertility. This project has been funded by the Australian National Health and Medical Research Council (G1600095) and The Hunter Medical Research Institute Bob and Terry Kennedy Children's Research Project Grant in Pregnancy & Reproduction (G1501433). All authors declare no conflict of interests.

Quiet sleep detection in preterm infants using deep convolutional neural networks

Objective. Neonates spend most of their time asleep. Sleep of preterm infants evolves rapidly throughout maturation and plays an important role in brain development. Since visual labelling of the sleep stages is a time consuming task, automated analysis of electroencephalography (EEG) to identify sleep stages is of great interest to clinicians. This automated sleep scoring can aid in optimizing neonatal care and assessing brain maturation. Approach. In this study, we designed and implemented an 18-layer convolutional neural network to discriminate quiet sleep from non-quiet sleep in preterm infants. The network is trained on 54 recordings from 13 preterm neonates and the performance is assessed on 43 recordings from 13 independent patients. All neonates had a normal neurodevelopmental outcome and the EEGs were recorded between 27 and 42 weeks postmenstrual age. Main results. The proposed network achieved an area under the mean and median ROC curve equal to 92% and 98%, respectively. Significance. Our findings suggest that CNN is a suitable and fast approach to classify neonatal sleep stages in preterm infants.

Proper timing for the evaluation of neonatal brain white matter development: a diffusion tensor imaging study.

We aimed to determine the timing for assessing birth status of the developing brain (i.e. brain maturity at birth) by exploring the postnatal age-related changes in neonatal brain white matter (WM). The institutional review board approved this study and all informed parental consents were obtained. 133 neonates (gestational age, 30-42 weeks) without abnormalities on MRI were studied with regard to WM development by diffusion tensor imaging-derived fractional anisotropy (FA). Tract-based spatial statistics (TBSS), locally-weighted scatterplot smoothing (LOESS) and piecewise linear-fitting were used to investigate the relationship between FA and postnatal age. FA along corticospinal tract (CST), optic radiation (OR), auditory radiation (AR) and thalamus-primary somatosensory cortex (thal-PSC) were extracted by automated fibre-tract quantification; their differences and associations with neonatal neurobehavioural scores at various postnatal age ranges were analysed by Wilcoxon's rank-sum test and Pearson's correlation. Using TBSS, postnatal age (days 1-28) positively correlated with FA in multiple WMs, including CST, OR, AR and thal-PSC (p<0.05). On the other hand, when narrowing the postnatal age window to days 1-14, no significant correlation was found, suggesting a biphasic WM development. LOESS and piecewise linear-fitting indicated that FA increased mildly before day 14 and its growth accelerated thereafter. Both FA and correlations with neurobehavioural scores in postnatal age range 2 (days 15-28) were significantly higher than in range 1 (days 1-14) (FA comparison: p<0.05; maximal correlation-coefficient: 0.693 vs. 0.169). Brain WM development during the neonatal stage includes two phases, i.e. a close-to-birth period within the first 14 days and a following accelerated maturation period. Therefore, evaluations of birth status should preferably be performed during the first period. • Brain white matter development within the first two postnatal weeks resembles a close-to-birth maturation. • Brain white matter development in the audio-visual, sensorimotor regions accelerates after two postnatal weeks. • Postnatal age-related effects should be considered in comparing preterm and term neonates.

CXCR3high CD8+ T cells with naïve phenotype and high capacity for IFN-γ production are generated during homeostatic T-cell proliferation.

Naïve phenotype (NP) Tcells spontaneously initiate homeostatic proliferation (HP) as T-cell output is reduced because of physiologic thymic involution with age. However, the effects of sustained HP on overall immune function are poorly understood. We demonstrated that the NP CD8+ Tcell population in adult thymectomized mice showing accelerated HP has an increased capacity for TCR-mediated interferon-γ and tumor necrosis factor α production, which is attributed to an increase in CXCR3+ cells in the NP CD8+ Tcell population. The CXCR3+ NP CD8+ Tcells developed during persistent HP with a slow cell division rate, but rarely during robust antigen-driven proliferation with a fast cell division rate. In ontogeny, the proportions of CXCR3+ cells in the NP CD8+ Tcell population showed a biphasic profile, which was high at the newborn and aged stages. Upon transfer, CXCR3+ NP CD8+ Tcells, but not CXCR3- NP CD8+ Tcells, potently enhanced Th17-mediated inflammatory tissue reactions in vivo. Furthermore, CXCR3high NP CD8+ Tcells with similar features were also detected at variable levels in healthy human blood. These results suggest that CXCR3+ NP CD8+ Tcells generated during physiological HP significantly impact overall immunity at the immunologically vulnerable neonatal and aged stages.

Abstract 308: Wls Inhibits Fibrosis Through Wnt Secretion From the Cardiomyocytes During Heart Regeneration

Because cardiomyocytes lose their capacity for self-renewal shortly after birth, the heart cannot regenerate muscle lost due to injury or heart failure. Instead, dead myocardium is replaced with a fibrotic scar. Recent studies have demonstrated that blocking the Hippo pathway specifically in cardiomyocytes promotes heart functional recovery after injury, not only by stimulating cardiomyocyte proliferation, but also by preventing fibrosis. The mechanism through which YAP activates the cell cycle is widely studied; however, the mechanism through which it inhibits fibrosis is unknown. Here, we identified Wntless ( Wls ) to be a downstream target of YAP in regenerating cardiomyocytes; Wls plays a role in regulating the secretion of Wnt ligands, which influences interstitial cells through paracrine effects. By using Adeno-Associated Virus 9 (AAV9) to deliver guide RNAs (gRNAs) that target the Wls locus exclusively in cardiomyocytes that express the Cas9 protein, we generated a cardiomyocyte-specific Wls knock out mouse (Wls-Cas9 CKO). After myocardial infarction at the neonatal stage (regenerative stage), cardiomyocyte deletion of Wls detrimental the heart regeneration as showed decreased heart function and increased fibrosis in Wls-Cas9 CKO mice. Mechanistically, by performing nuclear RNA-Seq technology, we revealed that non-canonical Wnt ligands are enriched in cardiomyocytes suggest cardiomyocytes influence fibroblasts through non-canonical signaling during heart regeneration. We will further use the single-cell sequencing technology to investigate the cell to cell communication during heart regeneration.

Vitamin A administration at birth promotes calf growth and intramuscular fat development in Angus beef cattle

BackgroundMarbling, or intramuscular fat, is an important factor contributing to the palatability of beef. Vitamin A, through its active metabolite, retinoic acid, promotes the formation of new fat cells (adipogenesis). As intramuscular adipogenesis is active during the neonatal stage, we hypothesized that vitamin A administration during the neonatal stage would enhance intramuscular adipogenesis and marbling.MethodsAngus steer calves (n = 30), in a completely randomized design, were randomly allotted to three treatment groups at birth, receiving 0, 150,000, or 300,000 IU of vitamin A at both birth and one month of age. A biopsy of the biceps femoris muscle was collected at two months of age. After weaning at 210 d of age, steers were fed a backgrounding diet in a feedlot until 308 d of age, when they were transitioned to a high concentrate finishing diet and implanted with trenbolone/estradiol/tylosin mixture. Steers were harvested at an average of 438 d of age. All diets were formulated to meet nutrient requirements.ResultsWeaning weight and weight during the backgrounding phase were linearly increased (P < 0.05) by vitamin A level, though no difference in body weight was observed at harvest. Intramuscular fat of steers at 308 d of age, measured by ultrasound, quadratically increased (P < 0.05) with vitamin A level from 4.0±0.26 % to 4.9±0.26 %. Similarly, carcass marbling score in the ribeye quadratically increased (P < 0.05).ConclusionAdministration of vitamin A at birth increased weaning weight and enhanced marbling fat development. Thus, vitamin A administration provides a practical method for increasing marbling and early growth of beef cattle.

Early Generated B-1-Derived B Cells Have the Capacity To Progress To Become Mantle Cell Lymphoma-like Neoplasia in Aged Mice.

In mice, fetal/neonatal B-1 cell development generates murine CD5+ B cells (B1a) with autoreactivity. We analyzed B1a cells at the neonatal stage in a VH11/D/JH knock-in mouse line (VH11t) that generates an autoreactive antiphosphatidylcholine BCR. Our study revealed that antiphosphatidylcholine B1a cells develop in liver, mature in spleen, and distribute in intestine/colon, mesenteric lymph node (mLN), and body cavity as the outcome of B-1 cell development before B-2 cell development. Throughout life, self-renewing B-1 B1a cells circulate through intestine, mesenteric vessel, and blood. The body cavity-deposited B1a cells also remigrate. In old age, some B1a cells proceed to monoclonal B cell lymphocytosis. When neonatal B-1 B1a cells express an antithymocyte/Thy-1 autoreactivity (ATA) BCR transgene in the C.B17 mouse background, ATA B cells increase in PBL and strongly develop lymphomas in aging mice that feature splenomegaly and mLN hyperplasia with heightened expression of CD11b, IL-10, and activated Stat3. At the adult stage, ATA B cells were normally present in the mantle zone area, including in intestine. Furthermore, frequent association with mLN hyperplasia suggests the influence by intestinal microenvironment on lymphoma development. When cyclin D1 was overexpressed by the Eμ-cyclin D1 transgene, ATA B cells progressed to further diffused lymphoma in aged mice, including in various lymph nodes with accumulation of IgMhiIgDloCD5+CD23-CD43+ cells, resembling aggressive human mantle cell lymphoma. Thus, our findings reveal that early generated B cells, as an outcome of B-1 cell development, can progress to become lymphocytosis, lymphoma, and mantle cell lymphoma-like neoplasia in aged mice.

Novel Spatiotemporal Analysis for Exploring a-Cell Differentiation

Aims: In vivo studies have demonstrated that pancreatic α-cells have potency to transdifferentiate into insulin-producing β cells, which could lead to potential therapy to cure diabetes. In addition, inappropriate glucagon secretion from α cells has been shown to be involved in the pathophysiology of diabetes. Although such findings highlight the need for better understanding of α-cell biology, it remains to be elucidated where α cells originate and what regulates α cell fate: α cell neogenesis, maturation and dedifferentiation. To address this issue, we generated a novel reporter mouse “Gcg-Timer.” Methods: We have previously generated Neurog3-Timer mouse (Miyatsuka et al. Diabetes 2009), in which newborn endocrine cells can be sorted by FACS separately from differentiated cells. However, we failed to obtain spatiotemporal information of newborn cells due to the low fluorescent intensity of DsRed-E5. To overcome this difficulty, we used a new reporter construct eGFP-IRES-mRFP and inserted it into the proglucagon gene. In this Gcg-Timer mouse, green fluorescence can be detected first, because mRFP expression is controlled by IRES and therefore lower than eGFP expression. As a result, newly-generated α cells exhibit green fluorescence and can be distinguished from more differentiated α cells exhibiting both green and red fluorescence. Results: Both microscopic analysis and flow cytometry revealed that green-dominant α cells were observed in Gcg-Timer mice at embryonic and neonatal stages until postnatal day 3 (P3), but not at P14 and adult stages. Intriguingly, red dominant cells, which were negative for glucagon staining, were detected at P14, indicating that α-to-non-α transition occurs at this stage. Conclusions: Gcg-Timer mice enabled us to label not only newly-generated α cells but also dedifferentiated α cells in a time-dependent manner. The α-cell neogenesis occurs specifically during embryonic and early neonatal stages under physiological condition. Disclosure M. Himuro: None. T. Miyatsuka: Research Support; Self; Eli Lilly and Company, Japan Society for the Promotion of Science, Daiichi Sankyo Company, Limited, Novartis Pharma K.K., Astellas Pharma Inc., MSD K.K., Takeda Pharmaceutical Company Limited.. L. Suzuki: None. M. Miura: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Kowa Pharmaceuticals America, Inc.. T. Katahira: None. M. Takahashi: None. Y. Nishida: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, AstraZeneca. S. Oshima: None. R. Okamoto: None. H. Watada: Advisory Panel; Self; AstraZeneca. Consultant; Self; Astellas Pharma US, Inc., AstraZeneca, Boehringer Ingelheim GmbH, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Novartis AG, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Development Center Asia, Pte. Ltd.. Research Support; Self; Abbott, Astellas Pharma US, Inc., AstraZeneca, Bayer AG, Benefit One Health Care Co., Ltd., Boehringer Ingelheim GmbH, Bristol-Myers Squibb Company, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Johnson &amp; Johnson Diabetes Institute, LLC., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nitto Boseki Co., Ltd., Novartis AG, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Terumo Medical Corporation.

Oral administration of Bifidobacterium bifidum TMC3115 to neonatal mice may alleviate IgE-mediated allergic risk in adulthood.

This study aimed to demonstrate whether exposure to bifidobacteria during early life influences immunity and alleviates the risk of immunoglobulin E (IgE)-mediated allergies in adulthood. BALB/c neonatal mice (n=54) were administered with a lyophilised cell preparation of Bifidobacterium bifidum TMC3115 (TMC3115) for 3 weeks. Following the intervention, the mice were immunised with intraperitoneal ovalbumin (OVA). The morphology and function of the intestinal epithelium were determined using histopathological examinations. Intestinal microbiota was detected using quantitative PCR and characterised using next-generation sequencing of 16S rRNA genes from faecal DNA. Caecal short-chain fatty acids (SCFAs) were measured using gas chromatography-mass spectrometry. Serum levels of tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and immunoglobulin E (IgE) and the percentage of splenic CD4+ T cells were examined using enzyme-linked immunosorbent assay and flow cytometry, respectively. TMC3115 did not significantly affect body weight, and cause any severe systemic inflammation or other clinical symptoms among the neonatal or adult mice, although the crypt depths and Muc2-positive cells in some intestinal segments of neonatal mice were significantly lower than control. Oral TMC3115 administration significantly increased faecal microbial diversity, relative abundance of Bacteroidetes and caecal SCFAs production in neonatal mice. Following the intervention, neonatal mice treated with TMC3115 exhibited less increase in serum IgE levels induced by OVA in adults and significantly higher TNF-α and IL-10 levels than in control. Our findings indicate that the oral administration of bifidobacteria, particularly certain strains, such as TMC3115, during early life could alleviate the risk of IgE-mediated allergies in adult host animals. Modifications of intestinal microbiota, SCFAs metabolism and anti-inflammatory cytokine IL-10 production by bifidobacteria may at least in part be a key mechanism underlying the effect of bifidobacteria on the IgE-mediated immune sensitivity of hosts to attacks by allergens at both neonatal and adult stages.

Nerve Injury-Induced Chronic Pain Is Associated with Persistent DNA Methylation Reprogramming in Dorsal Root Ganglion.

Nerve injury-induced hyperactivity of primary sensory neurons in the dorsal root ganglion (DRG) contributes to chronic pain development, but the underlying epigenetic mechanisms remain poorly understood. Here we determined genome-wide changes in DNA methylation in the nervous system in neuropathic pain. Spinal nerve ligation (SNL), but not paclitaxel treatment, in male Sprague Dawley rats induced a consistent low-level hypomethylation in the CpG sites in the DRG during the acute and chronic phases of neuropathic pain. DNA methylation remodeling in the DRG occurred early after SNL and persisted for at least 3 weeks. SNL caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands, in introns, intergenic regions, and repetitive sequences. In contrast, SNL caused more gains of methylation in the spinal cord and prefrontal cortex. The DNA methylation changes in the injured DRGs recapitulated developmental reprogramming at the neonatal stage. Methylation reprogramming was correlated with increased gene expression variability. A diet deficient in methyl donors induced hypomethylation and pain hypersensitivity. Intrathecal administration of the DNA methyltransferase inhibitor RG108 caused long-lasting pain hypersensitivity. DNA methylation reprogramming in the DRG thus contributes to nerve injury-induced chronic pain. Restoring DNA methylation may represent a new therapeutic approach to treat neuropathic pain.SIGNIFICANCE STATEMENT Epigenetic mechanisms are critically involved in the transition from acute to chronic pain after nerve injury. However, genome-wide changes in DNA methylation in the nervous system and their roles in neuropathic pain development remain unclear. Here we used digital restriction enzyme analysis of methylation to quantitatively determine genome-wide DNA methylation changes caused by nerve injury. We showed that nerve injury caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands in the dorsal root ganglion. Reducing DNA methylation induced pain hypersensitivity, whereas increasing DNA methylation attenuated neuropathic pain. These findings extend our understanding of the epigenetic mechanism of chronic neuropathic pain and suggest new strategies to treat nerve injury-induced chronic pain.

Incidence, risk factors, and outcome of neonatal acute kidney injury: a prospective cohort study.

Acute kidney injury (AKI) is common in neonates admitted to the neonatal intensive care unit (NICU). AKI is associated with increased morbidity and mortality and a greater long-term risk of chronic kidney disease. To study the incidence and outcome of neonatal AKI in a single Saudi Arabian center, level 2\3 NICU. This single-center prospective cohort study included all infants who received level II or III NICU care during 2015 (January to December). We excluded infants who survived less than 48h after admission, had evidence of congenital renal anomalies, or had insufficient data. AKI was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Both AKI and non-AKI neonates were prospectively followed up until NICU discharge. Outcomes studied included mortality and length of NICU stay. The results of adjusted risk analyses were expressed as relative risk (RR) with 95% confidence interval (95% CI). The incidence of AKI (modified neonatal KDIGO stages) was 56% (120/214 patients). Compared with neonates without AKI, those with AKI had a lower birth weight (2202 ± 816 vs. 1570 ± 776g; p < 0.001) and a lower gestational age (35 ± 3 vs. 32 ± 4weeks; p < 0.001). After adjustment for potential confounders, only gestational age (RR, 4.8; 95% CI, 3-9) and perinatal depression (RR, 10; 95% CI, 2-46) were significantly associated with an increased risk of AKI. For infants with gestational age < 32weeks, only the Clinical Risk Index for Babies (CRIB II) score was associated with an increased risk of AKI (RR, 1.9; 95% CI, 1-3). After adjustment for gestational age, AKI was significantly associated with mortality (RR, 5.4; 95% CI, 2-14), but not with the length of hospital stay (LOS) (p = 0.133). However, the AKI group had a significantly longer LOS (mean difference: 14days; 95% CI, 5.5-23days), and 33 patients (27.5%) with AKI were discharged with abnormally high serum creatinine levels (> 65μmol/L). AKI occurred in more than half of all NICU admissions, was associated with an increased risk of mortality, and had a higher incidence among smaller and sicker infants. Therefore, close monitoring of renal function during hospitalization and after discharge is warranted in such infants.