Eat, Sleep, Console Approach or Usual Care for Neonatal Opioid Withdrawal.

BACKGROUND: Although clinicians have traditionally used the Finnegan Neonatal Abstinence Scoring Tool to assess the severity of neonatal opioid withdrawal, a newer function-based approach - the Eat, Sleep, Console care approach - is increasing in use. Whether the new approach can safely reduce the time until infants are medically ready for discharge when it is applied broadly across diverse sites is unknown. METHODS: In this cluster-randomized, controlled trial at 26 U.S. hospitals, we enrolled infants with neonatal opioid withdrawal syndrome who had been born at 36 weeks' gestation or more. At a randomly assigned time, hospitals transitioned from usual care that used the Finnegan tool to the Eat, Sleep, Console approach. During a 3-month transition period, staff members at each hospital were trained to use the new approach. The primary outcome was the time from birth until medical readiness for discharge as defined by the trial. Composite safety outcomes that were assessed during the first 3 months of postnatal age included in-hospital safety, unscheduled health care visits, and nonaccidental trauma or death. RESULTS: A total of 1305 infants were enrolled. In an intention-to-treat analysis that included 837 infants who met the trial definition for medical readiness for discharge, the number of days from birth until readiness for hospital discharge was 8.2 in the Eat, Sleep, Console group and 14.9 in the usual-care group (adjusted mean difference, 6.7 days; 95% confidence interval [CI], 4.7 to 8.8), for a rate ratio of 0.55 (95% CI, 0.46 to 0.65; P<0.001). The incidence of adverse outcomes was similar in the two groups. CONCLUSIONS: As compared with usual care, use of the Eat, Sleep, Console care approach significantly decreased the number of days until infants with neonatal opioid withdrawal syndrome were medically ready for discharge, without increasing specified adverse outcomes. (Funded by the Helping End Addiction Long-term (HEAL) Initiative of the National Institutes of Health; ESC-NOW ClinicalTrials.gov number, NCT04057820.).

Placental Inflammation Significantly Correlates with Reduced Risk for Retinopathy of Prematurity.

Retinopathy of prematurity (ROP), a blinding condition affecting preterm infants, is an interruption of retinal vascular maturation that is incomplete when born preterm. Although ROP demonstrates delayed onset following preterm birth, representing a window for therapeutic intervention, there are no curative or preventative measures available for this condition. The in utero environment, including placental function, is increasingly recognized for contributions to preterm infant disease risk. The current study identified a protective association between acute placental inflammation and preterm infant ROP development using logistic regression, with the most significant association found for infants without gestational exposure to maternal preeclampsia and those with earlier preterm birth. Expression analysis of proteins with described ROP risk associations demonstrated significantly decreased placental high temperature requirement A serine peptidase-1 (HTRA-1) and fatty acid binding protein 4 protein expression in infants with acute placental inflammation compared with those without. Within the postnatal peripheral circulation, HTRA-1 and vascular endothelial growth factor-A demonstrated inverse longitudinal trends for infants born in the presence of, compared with absence of, acute placental inflammation. An agnostic approach, including whole transcriptome and differential methylation placental analysis, further identify novel mediators and pathways that may underly protection. Taken together, these data build on emerging literature showing a protective association between acute placental inflammation and ROP development and identify novel mechanisms that may inform postnatal risk associations in preterm infants.

Sexual Dimorphism in the Closure of the Hippocampal Postnatal Critical Period of Synaptic Plasticity after Intrauterine Growth Restriction: Link to Oligodendrocyte and Glial Dysregulation.

Intrauterine growth restriction (IUGR) resulting from hypertensive disease of pregnancy (HDP) leads to sexually dimorphic hippocampal-dependent cognitive and memory impairment in humans. In our translationally relevant mouse model of IUGR incited by HDP, we have previously shown that the synaptic development in the dorsal hippocampus including GABAergic development, NPTX2+ excitatory synaptic formation, axonal myelination, and perineural net (PNN) formation were perturbed by IUGR at adolescent equivalence in humans (P40). The persistence of these disturbances through early adulthood and the potential upstream mechanisms are currently unknown. Thus, we hypothesized that NPTX2+ expression, PNN formation, axonal myelination, all events closing synaptic development in the hippocampus, will be persistently perturbed, particularly affecting IUGR female mice through P60 given the fact that they had worse short-term recognition memory in this model. We additionally hypothesized that such sexual dimorphism is linked to persistent glial dysregulation. We induced IUGR by a micro-osmotic pump infusion of a potent vasoconstrictor U-46619, a thromboxane A2-analog, in the last week of the C57BL/6 mouse gestation to precipitate HDP. Sham-operated mice were used as controls. At P60, we assessed hippocampal and hemispheric volumes, NPTX2 expression, PNN formation, as well as myelin basic protein (MBP), Olig2, APC/CC1, and M-NF expression. We also evaluated P60 astrocytic (GFAP) reactivity and microglial (Iba1 and TMEM119) activation using immunofluorescent-immunohistochemistry and Imaris morphological analysis plus cytokine profiling using Meso Scale Discovery platform. IUGR offspring continued to have smaller hippocampal volumes at P60 not related to changes in hemisphere volume. NPTX2+ puncta counts and volumes were decreased in IUGR hippocampal CA subregions of female mice compared to sex-matched shams. Intriguingly, NPTX2+ counts and volumes were concurrently increased in the dentate gyrus (DG) subregion. PNN volumes were smaller in CA1 and CA3 of IUGR female mice along with PNN intensity in CA3 but they had larger volumes in the CA3 of IUGR male mice. The myelinated axon (MBP+) areas, volumes, and lengths were all decreased in the CA1 of IUGR female mice compared to sex-matched shams, which correlated with a decrease in Olig2 nuclear expression. No decrease in the number of APC/CC1+ mature oligodendrocytes was identified. We noted an increase in M-NF expression in the mossy fibers connecting DG to CA3 only in IUGR female mice. Reactive astrocytes denoted by GFAP areas, volumes, lengths, and numbers of branching were increased in IUGR female CA1 but not in IUGR male CA3 compared to sex-matched shams. Lastly, activated microglia were only detected in IUGR female CA1 and CA3 subregions. We detected no difference in the cytokine profile between sham and IUGR adult mice of either sex. Collectively, our data support a sexually dimorphic impaired closure of postnatal critical period of synaptic plasticity in the hippocampus of young adult IUGR mice with greater effects on females. A potential mechanism supporting such dimorphism may include oligodendrocyte dysfunction in IUGR females limiting myelination, allowing axonal overgrowth followed by a reactive glial-mediated synaptic pruning.

Retinopathy of prematurity protection conferred by uteroplacental insufficiency through erythropoietin signaling in an experimental Murine Model.

BACKGROUND: Recent clinical studies suggest that preeclampsia, characterized by uteroplacental insufficiency (UPI) and infant intrauterine growth restriction (IUGR), may be protective against retinopathy of prematurity (ROP) in preterm infants. Experimental models of UPI/IUGR have found an association of erythropoietin (EPO) with less severe oxygen-induced retinopathy (OIR); however, it is unclear if EPO/EPO receptor (EPOR) signaling was involved. We hypothesized that maternal UPI and resultant infant IUGR would protect against features of ROP through EPO/EPOR signaling. METHODS: We compared transgenic mice with hypoactive EPOR signaling (hWtEPOR) to littermate wild-type mice (mWtEpoR) in a novel combined model of IUGR and ROP. Thromboxane A2 (TXA2) was infused into pregnant C57Bl/6J dams to produce UPI/IUGR; postnatal pups and their foster dams were subjected to a murine OIR model. RESULTS: Following hyperoxia, hematocrits were similar between littermate wild-type (mWtEpoR) TXA2/OIR and vehicle/OIR pups. mWtEpoR TXA2/OIR had increased serum EPO, retinal EPO and VEGF, and decreased avascular retinal area (AVA) compared to vehicle/OIR pups. In comparison to the mWtEpoR TXA2/OIR pups, AVA was not reduced in hWtEPOR TXA2/OIR pups. CONCLUSION: Our findings provide biologic evidence that UPI/OIR-induced endogenous EPOR signaling confers protection against hyperoxia-induced vascular damage that may be related to pathophysiology in ROP. IMPACT: Maternal preeclampsia and infant growth restriction confer retinovascular protection against high oxygen-induced damage through endogenous erythropoietin signaling.

Intrauterine Growth Restriction Disrupts the Postnatal Critical Period of Synaptic Plasticity in the Mouse Dorsal Hippocampus in a Model of Hypertensive Disease of Pregnancy.

INTRODUCTION: Intrauterine growth restriction (IUGR) from hypertensive disease of pregnancy complicates up to 10% of all pregnancies. Significant hippocampal-dependent cognitive and memory impairments as well as neuropsychiatric disorders have been linked to IUGR. Because disturbance of the hippocampal critical period (CPd) of synaptic plasticity leads to impairments similar to those described in IUGR human offspring, we hypothesized that IUGR would perturb the CPd of synaptic plasticity in the mouse hippocampus in our model. METHODS: IUGR was produced by a micro-osmotic pump infusion of the potent vasoconstrictor U-46619, a thromboxane A2-agonist, at embryonic day 12.5 in C57BL/6J mouse dams to precipitate hypertensive disease of pregnancy and IUGR. Sham-operated mice acted as controls. At P10, P18, and P40, we assessed astrogliosis using GFAP-IHC. In dorsal CA1 and CA3 subfields, we assessed the immunoreactivities (IR) (IF-IHC) to (i) parvalbumin (PV) and glutamate decarboxylase (GAD) 65/67, involved in CPd onset; (ii) PSA-NCAM that antagonizes CPd onset; (iii) NPTX2, necessary for excitatory synapse formation and engagement of CPd; and (iv) MBP and WFA, staining perineural nets (PNNs), marking CPd closure. ImageJ/Fiji and IMARIS were used for image processing and SPSS v24 for statistical analysis. RESULTS: Although PV+ interneuron numbers and IR intensity were unchanged, development of GAD65/67+ synaptic boutons was accelerated at P18 IUGR mice and inversely correlated with decreased expression of PSA-NCAM in the CA of P18 IUGR mice at P18. NPTX2+ puncta and total volume were persistently decreased in the CA3 pyramidal and radiatum layers of IUGR mice from P18 to P40. At P40, axonal myelination (MBP+) in CA3 of IUGR mice was decreased and correlated with NPTX2 deficits. Lastly, the volume and integrity of the PNNs in the dorsal CA was disrupted in IUGR mice at P40. DISCUSSION/CONCLUSION: IUGR disrupts the molecular and structural initiation, consolidation, and closure of the CPd of synaptic plasticity in the mouse hippocampus in our model, which may explain the learning and memory deficits observed in juvenile IUGR mice and the cognitive disorders seen in human IUGR offspring. The mechanistic links warrant further investigation, to identify therapeutic targets to prevent neurodevelopmental deficits in patients affected by IUGR.

Hospital Outcomes of Infants with Neonatal Opioid Withdrawal Syndrome at a Tertiary Care Hospital with High Rates of Concurrent Nonopioid (Polysubstance) Exposure.

OBJECTIVE: Neonatal opioid withdrawal syndrome (NOWS) describes infants' withdrawal signs and symptoms after birth due to an interruption of prenatal opioid exposure. Many infants with NOWS are also exposed to nonopioids, however. This study was to determine hospital outcomes of infants exposed to opioids alone or coexposed with nonopioid substances (polysubstance). STUDY DESIGN: We reviewed infants of ≥34 weeks of gestation with prenatal opioid exposure from April 2015 to May 2018. We compared the median lengths of stay (LOS) and treatment (LOT) and the percentages of infants requiring pharmacologic and adjunctive treatment in infants exposed to opioids alone or polysubstance. We used Wilcoxon's test for continuous outcomes or Chi-squared test for categorical outcomes to determine statistical significance. We used multivariable regression model to calculate each drug category's estimates of adjusted mean ratios for LOS and LOT plus estimates of adjusted odds ratios for pharmacologic/adjunctive treatments. RESULTS: Of the 175 infants, 33 (19%) infants had opioid exposure alone. Opioid exposure included short- and/or long-acting opioids. A total of 142 (81%) had polysubstance exposure with 47% of mothers using nicotine products. We saw similar hospital outcomes between infants exposed to opioids alone or polysubstance; however, a higher percentage of infants with both short- and long-acting opioid exposure required pharmacologic treatment compared with either opioid alone. Focusing on individual drug categories, we detected differential hospital outcomes in which short-acting opioids decreased LOT, whereas long-acting opioids increased LOS, LOT, and need for pharmacologic and adjunctive treatment. Coexposure of opioids with stimulants decreased LOT and reduced need for adjunctive treatment. Coexposures with antidepressants increased LOT, while with antiepilepetics increased LOS. CONCLUSION: Because infants with NOWS often have coexposures to other nonopioid substances, appreciating the associated risks of individual or combination of drugs in modulating hospital outcomes may help counsel families on their infants' expected hospital course. KEY POINTS: · Hospital outcomes were similar between infants exposed to opioids alone or polysubstance including opioids.. · Infants with short- and long-acting opioids required pharmacologic treatment more often than either opioid alone.. · Differential hospital outcomes exist for various co-exposures of opioids with nonopioids..

Lef1-dependent hypothalamic neurogenesis inhibits anxiety.

While innate behaviors are conserved throughout the animal kingdom, it is unknown whether common signaling pathways regulate the development of neuronal populations mediating these behaviors in diverse organisms. Here, we demonstrate that the Wnt/ß-catenin effector Lef1 is required for the differentiation of anxiolytic hypothalamic neurons in zebrafish and mice, although the identity of Lef1-dependent genes and neurons differ between these 2 species. We further show that zebrafish and Drosophila have common Lef1-dependent gene expression in their respective neuroendocrine organs, consistent with a conserved pathway that has diverged in the mouse. Finally, orthologs of Lef1-dependent genes from both zebrafish and mouse show highly correlated hypothalamic expression in marmosets and humans, suggesting co-regulation of 2 parallel anxiolytic pathways in primates. These findings demonstrate that during evolution, a transcription factor can act through multiple mechanisms to generate a common behavioral output, and that Lef1 regulates circuit development that is fundamentally important for mediating anxiety in a wide variety of animal species.

Intrauterine Growth Restriction and Hyperoxia as a Cause of White Matter Injury.

Intrauterine growth restriction (IUGR) is estimated to occur in 5% of pregnancies, with placental insufficiency being the most common cause in developed countries. While it is known that white matter injury occurs in premature infants, the extent of IUGR on white matter injury is less defined in term infants. We used a novel murine model that utilizes a thromboxane A2 (TXA2) analog (U46619), a potent vasoconstrictor, to induce maternal hypertension and mimic human placental insufficiency-induced IUGR to study the white matter. We also investigated the role of hyperoxia as an additional risk factor for white matter injury, as IUGR infants are at increased risk of respiratory comorbidities leading to increased oxygen exposure. We found that TXA2 analog-induced IUGR results in white matter injury as demonstrated by altered myelin structure and changes in the oligodendroglial cell/oligodendrocyte population. In addition, our study demonstrates that hyperoxia exposure independently results in white matter perturbation. To our knowledge, this is the first study to report single and combined effects of IUGR with hyperoxia impacting the white matter and motor function. These results draw attention to the need for close monitoring of motor development in IUGR babies following hospital discharge as well as highlighting the importance of limiting, as clinically feasible, the degree of oxygen overexposure to potentially improve motor outcomes in this population of infants.

Effects of excess thromboxane A2 on placental development and nutrient transporters in a Mus musculus model of fetal growth restriction.

Hypertensive disease of pregnancy (HDP) with placental insufficiency is the most common cause of fetal growth restriction (FGR) in the developed world. Despite the known negative consequences of HDP both to the mother and fetus, little is known about the longitudinal placental changes that occur as HDP progresses in pregnancy. This is because longitudinal sampling of human placentae during each gestation is impossible. Therefore, using a mouse model of thromboxane A2-analog infusion to mimic human HDP in the last trimester, we calculated placental efficiencies based on fetal and placental weights; quantified spongiotrophoblast and labyrinth thicknesses and vascular density within these layers; examined whether hypoxia signaling pathway involving vascular endothelial growth factor A (VEGFA) and its receptors (VEGFR1, VEGFR2) and matrix metalloproteinases (MMPs) contributed to vascular change; and examined nutrient transporter abundance including glucose transporters 1 and 3 (GLUT1, GLUT3), neutral amino acid transporters 1, 2, and 4 (SNAT1, SNAT2, and SNAT4), fatty acid transporters 2 and 4 (FATP2, FATP4), and fatty acid translocase (CD36) from embryonic day 15.5 to 19 in a 20-day C57Bl/6J mouse gestation. We conclude that early-to-mid gestation hypertensive placentae show compensatory mechanisms to preserve fetal growth by increasing placental efficiencies and maintaining abundance of important nutrient transporters. As placental vascular network diminishes over late hypertension, placental efficiency diminishes and fetal growth fails. Neither hypoxia signaling pathway nor MMPs mediated the vascular diminution in this model. Hypertensive placentae surprisingly exhibit a sex-differential expression of nutrient transporters in late gestation despite showing fetal growth failure in both sexes.

Intrauterine growth restriction inhibits expression of eukaryotic elongation factor 2 kinase, a regulator of protein translation.

Nutrient deprivation suppresses protein synthesis by blocking peptide elongation. Transcriptional upregulation and activation of eukaryotic elongation factor 2 kinase (eEF2K) blocks peptide elongation by phosphorylating eukaryotic elongation factor 2. Previous studies examining placentas from intrauterine growth restricted (IUGR) newborn infants show decreased eEF2K expression and activity despite chronic nutrient deprivation. However, the effect of IUGR on hepatic eEF2K expression in the fetus is unknown. We, therefore, examined the transcriptional regulation of hepatic eEF2K gene expression in a Sprague-Dawley rat model of IUGR. We found decreased hepatic eEF2K mRNA and protein levels in IUGR offspring at birth compared with control, consistent with previous placental observations. Furthermore, the CpG island within the eEF2K promoter demonstrated increased methylation at a critical USF 1/2 transcription factor binding site. In vitro methylation of this binding site caused near complete loss of eEF2K promoter activity, designating this promoter as methylation sensitive. The eEF2K promotor in IUGR offspring also lost the protective histone covalent modifications associated with unmethylated CGIs. In addition, the +1 nucleosome was displaced 3' and RNA polymerase loading was reduced at the IUGR eEF2K promoter. Our findings provide evidence to explain why IUGR-induced chronic nutrient deprivation does not result in the upregulation of eEF2K gene transcription.

Intrauterine growth restriction perturbs nucleosome depletion at a growth hormone-responsive element in the mouse IGF-1 gene.

Intrauterine growth restriction (IUGR) is a common human pregnancy complication. IUGR offspring carry significant postnatal risk for early-onset metabolic syndrome, which is associated with persistent reduction in IGF-1 protein expression. We have previously shown that preadolescent IUGR male mice have decreased hepatic IGF-1 mRNA and circulating IGF-1 protein at postnatal day 21, the age when growth hormone (GH) normally upregulates hepatic IGF-1 expression. Here we studied nucleosome occupancy and CpG methylation at a putative growth hormone-responsive element in intron 2 (in2GHRE) of the hepatic IGF-1 gene in normal, sham-operated, and IUGR mice. Nucleosome occupancy and CpG methylation were determined in embryonic stem cells (ESCs) and in liver at postnatal days 14, 21, and 42. For CpG methylation, additional time points out to 2 yr were analyzed. We confirmed the putative mouse in2GHRE was GH-responsive, and in normal mice, a single nucleosome was displaced from the hepatic in2GHRE by postnatal day 21, which exposed two STAT5b DNA binding sites. Nucleosome displacement correlated with developmentally programmed CpG demethylation. Finally, IUGR significantly altered the nucleosome-depleted region (NDR) at the in2GHRE of IGF-1 on postnatal day 21, with either complete absence of the NDR or with a shifted NDR exposing only one of two STAT5b DNA binding sites. An NDR shift was also seen in offspring of sham-operated mothers. We conclude that prenatal insult such as IUGR or anesthesia/surgery could perturb the proper formation of a well-positioned NDR at the mouse hepatic IGF-1 in2GHRE necessary for transitioning to an open chromatin state.

IUGR prevents IGF-1 upregulation in juvenile male mice by perturbing postnatal IGF-1 chromatin remodeling.

BACKGROUND: Intrauterine growth restriction (IUGR) offspring with rapid catch-up growth are at increased risk for early obesity especially in males. Persistent insulin-like growth factor-1 (IGF-1) reduction is an important risk factor. Using a mouse model of maternal hypertension-induced IUGR, we examined IGF-1 levels, promoter DNA methylation, and histone H3 covalent modifications at birth (D1). We additionally investigated whether prenatal perturbations could reset at preadolescence (D21). METHODS: IUGR was induced via maternal thromboxane A2-analog infusion in mice. RESULTS: IUGR uniformly decreased D1 IGF-1 mRNA and protein levels with reduced promoter 1 (P1) transcription and increased P1 DNA methylation. IUGR males also had increased H3K4ac at exon 5 and 3' distal UTR. At D21, IUGR males continued to have decreased IGF-1 levels, originating from both P1 and P2 with reduced 1A variant. IUGR males also had decreased activation mark of H3K4me3 at P1 compared with sham males. In contrast, D21 IUGR females normalized their IGF-1 levels, in association with an increased activation mark of H3K4me3 at P1 compared with sham females. CONCLUSION: IUGR uniformly affected D1 hepatic IGF-1 epigenetic modifications in both sexes. However, at preadolescence, IUGR males are unable to correct for the prenatal reduction possibly due to a more perturbed IGF-1 chromatin structure.

Maternal tobacco smoke increased visceral adiposity and serum corticosterone levels in adult male rat offspring.

BACKGROUND: Maternal tobacco smoke (MTS) predisposes human and rat offspring to visceral obesity in early adulthood. Glucocorticoid excess also causes visceral obesity. We hypothesized that in utero MTS would increase visceral adiposity and alter the glucocorticoid pathway in young adult rats. METHODS: We developed a novel model of in utero MTS exposure in pregnant rats by exposing them to cigarette smoke from E11.5 to term. Neonatal rats were cross-fostered to control dams and weaned to standard rat chow through young adulthood (postnatal day 60). RESULTS: We demonstrated increased visceral adiposity (193%)*, increased visceral adipose 11-ß hydroxysteroid dehydrogenase 1 mRNA (204%)*, increased serum corticosterone (147%)*, and no change in glucocorticoid receptor protein in adult male MTS rat offspring. Female rats exposed to MTS in utero demonstrated no change in visceral or subcutaneous adiposity, decreased serum corticosterone (60%)*, and decreased adipose glucocorticoid receptor protein (66%)*. *P < 0.05. CONCLUSION: We conclude that in utero MTS exposure increased visceral adiposity and altered in the glucocorticoid pathway in a sex-specific manner. We speculate that in utero MTS exposure programs adipose dysfunction in adult male rat offspring via alteration in the glucocorticoid pathway.

Influence of Eat, Sleep, and Console on Infants Pharmacologically Treated for Opioid Withdrawal: A Post Hoc Subgroup Analysis of the ESC-NOW Randomized Clinical Trial.

Importance: The function-based eat, sleep, console (ESC) care approach substantially reduces the proportion of infants who receive pharmacologic treatment for neonatal opioid withdrawal syndrome (NOWS). This reduction has led to concerns for increased postnatal opioid exposure in infants who receive pharmacologic treatment. However, the effect of the ESC care approach on hospital outcomes for infants pharmacologically treated for NOWS is currently unknown. Objective: To evaluate differences in opioid exposure and total length of hospital stay (LOS) for pharmacologically treated infants managed with the ESC care approach vs usual care with the Finnegan tool. Design, Setting, and Participants: This post hoc subgroup analysis involved infants pharmacologically treated in ESC-NOW, a stepped-wedge cluster randomized clinical trial conducted at 26 US hospitals. Hospitals maintained pretrial practices for pharmacologic treatment, including opioid type, scheduled opioid dosing, and use of adjuvant medications. Infants were born at 36 weeks' gestation or later, had evidence of antenatal opioid exposure, and received opioid treatment for NOWS between September 2020 and March 2022. Data were analyzed from November 2022 to January 2024. Exposure: Opioid treatment for NOWS and the ESC care approach. Main Outcomes and Measures: For each outcome (total opioid exposure, peak opioid dose, time from birth to initiation of first opioid dose, length of opioid treatment, and LOS), we used generalized linear mixed models to adjust for the stepped-wedge design and maternal and infant characteristics. Results: In the ESC-NOW trial, 463 of 1305 infants were pharmacologically treated (143/603 [23.7%] in the ESC care approach group and 320/702 [45.6%] in the usual care group). Mean total opioid exposure was lower in the ESC care approach group with an absolute difference of 4.1 morphine milligram equivalents per kilogram (MME/kg) (95% CI, 1.3-7.0) when compared with usual care (4.8 MME/kg vs 8.9 MME/kg, respectively; P = .001). Mean time from birth to initiation of pharmacologic treatment was 22.4 hours (95% CI, 7.1-37.7) longer with the ESC care approach vs usual care (75.4 vs 53.0 hours, respectively; P = .002). No significant difference in mean peak opioid dose was observed between groups (ESC care approach, 0.147 MME/kg, vs usual care, 0.126 MME/kg). The mean length of treatment was 6.3 days shorter (95% CI, 3.0-9.6) in the ESC care approach group vs usual care group (11.8 vs 18.1 days, respectively; P < .001), and mean LOS was 6.2 days shorter (95% CI, 3.0-9.4) with the ESC care approach than with usual care (16.7 vs 22.9 days, respectively; P < .001). Conclusion and Relevance: When compared with usual care, the ESC care approach was associated with less opioid exposure and shorter LOS for infants pharmacologically treated for NOWS. The ESC care approach was not associated with a higher peak opioid dose, although pharmacologic treatment was typically initiated later. Trial Registration: ClinicalTrials.gov Identifier: NCT04057820.

Effects of intrauterine growth restriction on embryonic hippocampal dentate gyrus neurogenesis and postnatal critical period of synaptic plasticity that govern learning and memory function.

Intrauterine growth restriction (IUGR) complicates up to 10% of human pregnancies and is the second leading cause of perinatal morbidity and mortality after prematurity. The most common etiology of IUGR in developed countries is uteroplacental insufficiency (UPI). For survivors of IUGR pregnancies, long-term studies consistently show a fivefold increased risk for impaired cognition including learning and memory deficits. Among these, only a few human studies have highlighted sex differences with males and females having differing susceptibilities to different impairments. Moreover, it is well established from brain magnetic resonance imaging that IUGR affects both white and gray matter. The hippocampus, composed of the dentate gyrus (DG) and cornu ammonis (CA) subregions, is an important gray matter structure critical to learning and memory, and is particularly vulnerable to the chronic hypoxic-ischemic effects of UPI. Decreased hippocampal volume is a strong predictor for learning and memory deficits. Decreased neuron number and attenuated dendritic and axonal morphologies in both the DG and CA are additionally seen in animal models. What is largely unexplored is the prenatal changes that predispose an IUGR offspring to postnatal learning and memory deficits. This lack of knowledge will continue to hinder the design of future therapy to improve learning and memory. In this review, we will first present the clinical susceptibilities and human epidemiology data regarding the neurological sequelae after IUGR. We will follow with data generated using our laboratory's mouse model of IUGR, that mimics the human IUGR phenotype, to dissect at the cellular and molecular alterations in embryonic hippocampal DG neurogenesis. We will lastly present a newer topic of postnatal neuron development, namely the critical period of synaptic plasticity that is crucial in achieving an excitatory/inhibitory balance in the developing brain. To our knowledge, these findings are the first to describe the prenatal changes that lead to an alteration in postnatal hippocampal excitatory/inhibitory imbalance, a mechanism that is now recognized to be a cause of neurocognitive/neuropsychiatric disorders in at-risk individuals. Studies are ongoing in our laboratory to elucidate additional mechanisms that underlie IUGR-induced learning and memory impairment and to design therapy aimed at ameliorating such impairment.

Unbiased Quantitative Single-Cell Morphometric Analysis to Identify Microglia Reactivity in Developmental Brain Injury.

Microglia morphological studies have been limited to the process of reviewing the most common characteristics of a group of cells to conclude the likelihood of a "pathological" milieu. We have developed an Imaris-software-based analytical pipeline to address selection and operator biases, enabling use of highly reproducible machine-learning algorithms to quantify at single-cell resolution differences between groups. We hypothesized that this analytical pipeline improved our ability to detect subtle yet important differences between groups. Thus, we studied the temporal changes in Iba1+ microglia-like cell (MCL) populations in the CA1 between P10-P11 and P18-P19 in response to intrauterine growth restriction (IUGR) at E12.5 in mice, chorioamnionitis (chorio) at E18 in rats and neonatal hypoxia-ischemia (HI) at P10 in mice. Sholl and convex hull analyses differentiate stages of maturation of Iba1+ MLCs. At P10-P11, IUGR or HI MLCs were more prominently 'ameboid', while chorio MLCs were hyper-ramified compared to sham. At P18-P19, HI MLCs remained persistently 'ameboid' to 'transitional'. Thus, we conclude that this unbiased analytical pipeline, which can be adjusted to other brain cells (i.e., astrocytes), improves sensitivity to detect previously elusive morphological changes known to promote specific inflammatory milieu and lead to worse outcomes and therapeutic responses.

Uteroplacental insufficiency alters rat hippocampal cellular phenotype in conjunction with ErbB receptor expression.

INTRODUCTION: Uteroplacental insufficiency (UPI) produces significant neurodevelopmental deficits affecting the hippocampus of intrauterine growth-restricted (IUGR) offspring. IUGR males have worse deficits as compared with IUGR females. The exact mechanisms underlying these deficits are unclear. Alterations in hippocampal cellular composition along with altered expression of neural stem cell (NSC) differentiation molecules may underlie these deficits. We hypothesized that IUGR hippocampi would be endowed with altered neuronal, astrocytic, and immature oligodendrocytic proportions at birth, with males showing greater cellular deficits. We further hypothesized that UPI would perturb rat hippocampal expression of ErbB receptors (ErbB-Rs) and neuregulin 1 (NRG1) at birth and at weaning to account for the short- and long-term IUGR neurological sequelae. METHODS: A well-established rat model of bilateral uterine artery ligation at embryonic day 19.5 was used to induce IUGR. RESULTS: As compared with gender-matched controls, IUGR offspring have altered hippocampal neuronal, astrocytic, and immature oligodendrocytic composition in a subregion- and gender-specific manner at birth. In addition, IUGR hippocampi have altered receptor type- and gender-specific ErbB-R expression at birth and at weaning. DISCUSSION: These cellular and molecular alterations may account for the neurodevelopmental complications of IUGR and for the male susceptibility to worse neurologic outcomes.

Short- and Long-Term Implications of Small for Gestational Age.

Fetal growth restriction (FGR) describes a fetus' inability to attain adequate weight gain based on genetic potential and gestational age and is the second most common cause of perinatal morbidity and mortality after prematurity. Infants who have suffered fetal growth restriction are at the greatest risks for short- and long-term complications. This article specifically details the neurologic and cardiometabolic sequalae associated with fetal growth restriction, as well as the purported mechanisms that underlie their pathogenesis. We end with a brief discussion about further work that is needed to gain a more complete understanding of fetal growth restriction.

Intrauterine growth restriction followed by oxygen support uniquely interferes with genetic regulators of myelination.

Intrauterine growth restriction (IUGR) and oxygen exposure in isolation and combination adversely affect the developing brain, putting infants at risk for neurodevelopmental disability including cerebral palsy. Rodent models of IUGR and postnatal hyperoxia have demonstrated oligodendroglial injury with subsequent white matter injury (WMI) and motor dysfunction. Here we investigate transcriptomic dysregulation in IUGR with and without hyperoxia exposure to account for the abnormal brain structure and function previously documented. We performed RNA sequencing and analysis using a mouse model of IUGR and found that IUGR, hyperoxia, and the combination of IUGR with hyperoxia (IUGR/hyperoxia) produced distinct changes in gene expression. IUGR in isolation demonstrated the fewest differentially expressed genes compared to control. In contrast, we detected several gene alterations in IUGR/hyperoxia; genes involved in myelination were strikingly downregulated. We also identified changes to specific regulators including TCF7L2, BDNF, SOX2, and DGCR8, through Ingenuity Pathway Analysis, that may contribute to impaired myelination in IUGR/hyperoxia. Our findings show that IUGR with hyperoxia induces unique transcriptional changes in the developing brain. These indicate mechanisms for increased risk for WMI in IUGR infants exposed to oxygen and suggest potential therapeutic targets to improve motor outcomes.Significance StatementThis study demonstrates that perinatal exposures of IUGR and/or postnatal hyperoxia result in distinct transcriptomic changes in the developing brain. In particular, we found that genes involved in normal developmental myelination, myelin maintenance, and remyelination were most dysregulated when IUGR was combined with hyperoxia. Understanding how multiple risk factors lead to WMI is the first step in developing future therapeutic interventions. Additionally, because oxygen exposure is often unavoidable after birth, an understanding of gene perturbations in this setting will increase our awareness of the need for tight control of oxygen use to minimize future motor disability.

Challenges in Implementing Exclusive Human Milk Diet to Extremely Low-Birth-Weight Infants in a Level III Neonatal Intensive Care Unit.

PROBLEM: Extremely low-birth-weight (ELBW) infants require fortification of human milk (HM) to prevent growth failure. Bovine milk-based fortifiers (BOV-f) may be associated with feeding intolerance and necrotizing enterocolitis. Evidence suggests that an exclusive HM diet (EHMD) using HM-based fortifier (HM-f) may improve these outcomes. INTERVENTION: EHMD was introduced as a quality improvement project to improve feeding tolerance in ELBW infants. METHOD: Implementation included establishing EHMD feeding protocol and growth monitoring. We compared infants receiving HM with BOV-f (n = 49) with infants receiving an EHMD (n = 15). The primary outcome was a reduction of no oral intake days due to feeding intolerance. RESULTS: The EHMD cohort had a more advanced GA (28 vs 26 weeks; P = .03), more males (66.7% vs 42.9%; P = .02), and higher incidence of SGA (40.0% vs 18.4%; P = .16) compared with the HM-f group. The EHMD cohort had fewer days with no oral intake (2 vs 5; P < .005), which is insignificant when adjusted for small for gestational age (SGA) (P = .26). The EHMD cohort vs. the HM-f cohort had a significant decrease in weight and length z-scores from birth to discharge (-1.09 vs -0.26 [P = .002]; -1.76 vs -0.83 [P = .02]). Inadequate weight gain persisted after adjustment for SGA. Interventions were performed to increase caloric intake and institute milk preparation changes. CONCLUSION: EHMD improved feeding tolerance in our ELBW infants. Observed growth failure might be skewed by SGA prevalence. We highlight that implementation of EHMD requires close growth assessment, especially for SGA infants.