Bacteria - derived short chain fatty acids restore sympathoadrenal responsiveness to hypoglycemia after antibiotic-induced gut microbiota depletion.

The microbiome co-evolved with their mammalian host over thousands of years. This commensal relationship serves a pivotal role in various metabolic, physiological, and immunological processes. Recently we discovered impaired adrenal catecholamine stress responses in germ-free mice suggesting developmental modification of the reflex arc or absence of an ongoing microbiome signal. To determine whether maturational arrest or an absent bacteria-derived metabolite was the cause, we tested whether depleting gut microbiome in young adult animals could also alter the peripheral stress responses to insulin-induced hypoglycemia. Groups of C57Bl6 male mice were given regular water (control) or a cocktail of non-absorbable broad-spectrum antibiotics (Abx) in the drinking water for two weeks before injection with insulin or saline. Abx mice displayed a profound decrease in microbial diversity and abundance of Bacteroidetes and Firmicutes, plus a markedly enlarged caecum and no detectable by-products of bacterial fermentation (sp. short chain fatty acids, SCFA). Tonic and stress-induced epinephrine levels were attenuated. Recolonization (Abx + R) restored bacterial diversity, but not the sympathoadrenal system responsiveness or caecal acetate, propionate and butyrate levels. In contrast, corticosterone (HPA) and glucagon (parasympathetic) resting values and responses to hypoglycemia remained similar across all conditions. Oral supplementation with SCFA improved epinephrine responses to hypoglycaemia. Whole genome shotgun sequence profiling of fecal samples from control, Abx and Abx + R cohorts identified nine microbes (SCFA producers) absent from both Abx and Abx + R groups. These results implicate gut microbiome depletion plus its attendant reduction in SCFA signalling in adversely affecting the release of epinephrine in response to hypoglycemia. We speculate that regardless of postnatal age, a mutable microbiome messaging system exists throughout life. Unravelling these mechanisms could lead to new therapeutic possibilities through controlled manipulation of the gut microbiota and its ability to alter systemic neurotransmitter responsiveness.

Implementing a Hospital-Based Safe Sleep Program for Newborns and Infants.

BACKGROUND: An unsafe sleep environment remains the leading contributor to unexpected infant death. PURPOSE: To determine the effectiveness of a quality improvement initiative developed to create a hospital-based safe sleep environment for all newborns and infants. METHODS: A multidisciplinary team from the well-baby nursery (WBN) and neonatal intensive care unit (NICU) of a 149-bed academic, quaternary care, regional referral center developed and implemented safe sleep environments within the hospital for all prior to discharge. To monitor compliance, the following were tracked monthly: documentation of parent education, caregiver surveys, and hospital crib check audits. On the inpatient general pediatric units, only hospital crib check audits were tracked. Investigators used Plan-Do-Study-Act (PDSA) cycles to evaluate the impact of the initiative from October 2015 through February 2018. RESULTS: Safe sleep education was documented for all randomly checked records (n = 440). A survey (n = 348) revealed that almost all caregivers (95.4%) reported receiving information on safe infant sleep. Initial compliance with all criteria in WBN (n = 281), NICU (n = 285), and general pediatric inpatient units (n = 121) was 0%, 0%, and 8.3%, respectively. At 29 months, WBN and NICU compliance with all criteria was 90% and 100%, respectively. At 7 months, general pediatric inpatient units' compliance with all criteria was 20%. IMPLICATIONS FOR PRACTICE: WBN, NICU and general pediatric inpatient unit collaboration with content experts led to unit-specific strategies that improved safe sleep practices. IMPLICATIONS FOR RESEARCH: Future studies on the impact of such an initiative at other hospitals are needed.

Effect of High-Dose Erythropoietin on Blood Transfusions in Extremely Low Gestational Age Neonates: Post Hoc Analysis of a Randomized Clinical Trial.

Importance: Extremely preterm infants are among the populations receiving the highest levels of transfusions. Erythropoietin has not been recommended for premature infants because most studies have not demonstrated a decrease in donor exposure. Objectives: To determine whether high-dose erythropoietin given within 24 hours of birth through postmenstrual age of 32 completed weeks will decrease the need for blood transfusions. Design, Setting, and Participants: The Preterm Erythropoietin Neuroprotection Trial (PENUT) is a randomized, double-masked clinical trial with participants enrolled at 19 sites consisting of 30 neonatal intensive care units across the United States. Participants were born at a gestational age of 24 weeks (0-6 days) to 27 weeks (6-7 days). Exclusion criteria included conditions known to affect neurodevelopmental outcomes. Of 3266 patients screened, 2325 were excluded, and 941 were enrolled and randomized to erythropoietin (n = 477) or placebo (n = 464). Data were collected from December 12, 2013, to February 25, 2019, and analyzed from March 1 to June 15, 2019. Interventions: In this post hoc analysis, erythropoietin, 1000 U/kg, or placebo was given every 48 hours for 6 doses, followed by 400 U/kg or sham injections 3 times a week through postmenstrual age of 32 weeks. Main Outcomes and Measures: Need for transfusion, transfusion numbers and volume, number of donor exposures, and lowest daily hematocrit level are presented herein. Results: A total of 936 patients (488 male [52.1%]) were included in the analysis, with a mean (SD) gestational age of 25.6 (1.2) weeks and mean (SD) birth weight of 799 (189) g. Erythropoietin treatment (vs placebo) decreased the number of transfusions (unadjusted mean [SD], 3.5 [4.0] vs 5.2 [4.4]), with a relative rate (RR) of 0.66 (95% CI, 0.59-0.75); the cumulative transfused volume (mean [SD], 47.6 [60.4] vs 76.3 [68.2] mL), with a mean difference of -25.7 (95% CI, 18.1-33.3) mL; and donor exposure (mean [SD], 1.6 [1.7] vs 2.4 [2.0]), with an RR of 0.67 (95% CI, 0.58-0.77). Despite fewer transfusions, erythropoietin-treated infants tended to have higher hematocrit levels than placebo-treated infants, most noticeable at gestational week 33 in infants with a gestational age of 27 weeks (mean [SD] hematocrit level in erythropoietin-treated vs placebo-treated cohorts, 36.9% [5.5%] vs 30.4% [4.6%] (P < .001). Of 936 infants, 160 (17.1%) remained transfusion free at the end of 12 postnatal weeks, including 43 in the placebo group and 117 in the erythropoietin group (P < .001). Conclusions and Relevance: These findings suggest that high-dose erythropoietin as used in the PENUT protocol was effective in reducing transfusion needs in this population of extremely preterm infants. Trial Registration: ClinicalTrials.gov Identifier: NCT01378273.

Presence of neonatal intensive care services at birth hospital and early intervention enrollment in infants ≤1500 g.

Objectives To determine whether the receipt of therapeutic services of very-low-birth-weight (VLBW; ≤1500 g) neonates inadvertently delivered at community Level 2 and 3 neonatal intensive care units (NICUs) compared with those born at a well-baby nursery (WBN; Level 1) differed. Methods This is a retrospective study of neonates who were born at Level 1 (WBN), 2, 3, and 4 NICUs and discharged from a Level 4 hospital (n = 529). All infants were evaluated at the Regional Neonatal Follow-up Program at 12 ± 1 months corrected gestational age (CA) and assessed for use of therapeutic services including: early intervention (EI), occupational therapy (OT), physical therapy (PT), speech therapy (ST), and special education (SE). Results Compared to infants born at community Level 2 and 3 NICU hospitals, those outborn at a community Level 1 WBN had significantly higher utilization of EI (90% vs. 62%) and PT (83% vs. 61%) at 12 months CA. This association persisted when controlling for covariates. Infants who required EI had significantly lower Bayley-III cognitive scores at 3 years of age. Conclusion VLBW infants outborn at WBN (Level 1) hospitals required more outpatient therapeutic services than those born at hospitals with NICU facilities. These results suggest that delivering at the appropriate community hospital level of care might be advantageous for long-term outcomes.

A Randomized Trial of Erythropoietin for Neuroprotection in Preterm Infants.

BACKGROUND: High-dose erythropoietin has been shown to have a neuroprotective effect in preclinical models of neonatal brain injury, and phase 2 trials have suggested possible efficacy; however, the benefits and safety of this therapy in extremely preterm infants have not been established. METHODS: In this multicenter, randomized, double-blind trial of high-dose erythropoietin, we assigned 941 infants who were born at 24 weeks 0 days to 27 weeks 6 days of gestation to receive erythropoietin or placebo within 24 hours after birth. Erythropoietin was administered intravenously at a dose of 1000 U per kilogram of body weight every 48 hours for a total of six doses, followed by a maintenance dose of 400 U per kilogram three times per week by subcutaneous injection through 32 completed weeks of postmenstrual age. Placebo was administered as intravenous saline followed by sham injections. The primary outcome was death or severe neurodevelopmental impairment at 22 to 26 months of postmenstrual age. Severe neurodevelopmental impairment was defined as severe cerebral palsy or a composite motor or composite cognitive score of less than 70 (which corresponds to 2 SD below the mean, with higher scores indicating better performance) on the Bayley Scales of Infant and Toddler Development, third edition. RESULTS: A total of 741 infants were included in the per-protocol efficacy analysis: 376 received erythropoietin and 365 received placebo. There was no significant difference between the erythropoietin group and the placebo group in the incidence of death or severe neurodevelopmental impairment at 2 years of age (97 children [26%] vs. 94 children [26%]; relative risk, 1.03; 95% confidence interval, 0.81 to 1.32; P = 0.80). There were no significant differences between the groups in the rates of retinopathy of prematurity, intracranial hemorrhage, sepsis, necrotizing enterocolitis, bronchopulmonary dysplasia, or death or in the frequency of serious adverse events. CONCLUSIONS: High-dose erythropoietin treatment administered to extremely preterm infants from 24 hours after birth through 32 weeks of postmenstrual age did not result in a lower risk of severe neurodevelopmental impairment or death at 2 years of age. (Funded by the National Institute of Neurological Disorders and Stroke; PENUT ClinicalTrials.gov number, NCT01378273.).

Postnatal glucocorticoid-induced hypomyelination, gliosis, and neurologic deficits are dose-dependent, preparation-specific, and reversible.

Postnatal glucocorticoids (GCs) are widely used in the prevention of chronic lung disease in premature infants. Their pharmacologic use is associated with neurodevelopmental delay and cerebral palsy. However, the effect of GC dose and preparation (dexamethasone versus betamethasone) on short and long-term neurological outcomes remains undetermined, and the mechanisms of GC-induced brain injury are unclear. We hypothesized that postnatal GC would induce hypomyelination and motor impairment in a preparation- and dose-specific manner, and that GC receptor (GR) inhibition might restore myelination and neurological function in GC-treated animals. Additionally, GC-induced hypomyelination and neurological deficit might be transient. To test our hypotheses, we treated prematurely delivered rabbit pups with high (0.5mg/kg/day) or low (0.2mg/kg/day) doses of dexamethasone or betamethasone. Myelin basic protein (MBP), oligodendrocyte proliferation and maturation, astrocytes, transcriptomic profile, and neurobehavioral functions were evaluated. We found that high-dose GC treatment, but not low-dose, reduced MBP expression and impaired motor function at postnatal day 14. High-dose dexamethasone induced astrogliosis, betamethasone did not. Mifepristone, a GR antagonist, reversed dexamethasone-induced myelination, but not astrogliosis. Both GCs inhibited oligodendrocyte proliferation and maturation. Moreover, high-dose dexamethasone altered genes associated with myelination, cell-cycle, GR, and mitogen-activated protein kinase. Importantly, GC-induced hypomyelination, gliosis, and motor-deficit, observed at day 14, completely recovered by day 21. Hence, high-dose, but not low-dose, postnatal GC causes reversible reductions in myelination and motor functions. GC treatment induces hypomyelination by GR-dependent genomic mechanisms, but astrogliosis by non-genomic mechanisms. GC-induced motor impairment and neurodevelopmental delay might be transient and recover spontaneously in premature infants.

Partial blockade of nicotinic acetylcholine receptors improves the counterregulatory response to hypoglycemia in recurrently hypoglycemic rats.

Recurrent exposure to hypoglycemia can impair the normal counterregulatory hormonal responses that guard against hypoglycemia, leading to hypoglycemia unawareness. This pathological condition known as hypoglycemia-associated autonomic failure (HAAF) is the main adverse consequence that prevents individuals with type 1 diabetes mellitus from attaining the long-term health benefits of tight glycemic control. The underlying molecular mechanisms responsible for the progressive loss of the epinephrine response to subsequent bouts of hypoglycemia, a hallmark sign of HAAF, are largely unknown. Normally, hypoglycemia triggers both the release and biosynthesis of epinephrine through activation of nicotinic acetylcholine receptors (nAChR) on the adrenal glands. We hypothesize that excessive cholinergic stimulation may contribute to impaired counterregulation. Here, we tested whether administration of the nAChR partial agonist cytisine to reduce postganglionic synaptic activity can preserve the counterregulatory hormone responses in an animal model of HAAF. Compared with nicotine, cytisine has limited efficacy to activate nAChRs and stimulate epinephrine release and synthesis. We evaluated adrenal catecholamine production and secretion in nondiabetic rats subjected to two daily episodes of hypoglycemia for 3 days, followed by a hyperinsulinemic hypoglycemic clamp on day 4. Recurrent hypoglycemia decreased epinephrine responses, and this was associated with suppressed TH mRNA induction (a measure of adrenal catecholamine synthetic capacity). Treatment with cytisine improved glucagon responses as well as epinephrine release and production in recurrently hypoglycemic animals. These data suggest that pharmacological manipulation of ganglionic nAChRs may be promising as a translational adjunctive therapy to avoid HAAF in type 1 diabetes mellitus.

Thyroid hormone supplementation in preterm infants born before 28 weeks gestational age and neurodevelopmental outcome at age 36 months.

BACKGROUND: Thyroid hormones are required for normal brain maturation, and neonatal plasma thyroid hormone concentrations are low in infants less than 28 weeks gestation. It is not known whether treatment of such infants with thyroid hormone improves neurodevelopmental outcome. METHODS: At three years corrected age, mental, motor, and neurological development was assessed in infants born at less than 28 weeks gestational age who had participated in a phase 1 trial of differing doses and modes of administration of thyroid hormone. The trial's endpoints were thyroid hormone (thyroxine, T4) and thyotropin plasma concentrations in eight study arms: six treated with T4 [4, 8, and 16 µg/(kg · day)], bolus or continuous], one treated with iodine only, and one treated with placebo. Follow-up at three years was not part of the original study goals. Developmental index scores, rates of cerebral palsy (CP), and rates of adverse outcome (death or moderate to severe delay in development and/or disabling CP) were compared between the eight study groups and between groups combined by dosage level, and between infants with and without T4 supplementation. RESULTS: Of 166 randomized infants, 32 (19%) died in the neonatal period. Of the 134 survivors, follow-up results were available for 89 children (66%). Mental and motor development and rates of cerebral palsy did not differ in any of the comparisons made. CONCLUSION: In this study, no differences in neurodevelopment were found in relation to thyroid hormone treatment, but power was insufficient to detect any but very large differences.

Hemophagocytic lymphohistiocytosis associated with necrotizing enterocolitis in premature newborns.

Hemophagocytic lymphohistiocytosis (HLH) is a systemic disease resulting from the excessive release of inflammatory cytokines by macrophages under prolonged antigenic stimulation. If untreated, it leads to multiorgan failure and death. Necrotizing enterocolitis (NEC) has not previously been associated with HLH. Here we report four preterm infants who were diagnosed with HLH associated with NEC. Two patients received chemotherapy and one survived. The other two infants succumbed to multiorgan failure. These results suggest that NEC may be a common clinical manifestation of HLH in premature neonates.

Stability of thyroid hormones during continuous infusion.

We investigated the stability of thyroid hormones during a mode of continuous drug infusion via polypropylene tubing using the same conditions that would be applied to treating patients in a hospital setting. The diluted thyroid hormones were prepared using aseptic technique, stored at 2-8°C (36-46°F) and tested within 24 h of preparation for stability and percent recovery from within plastic tubing. Experiments were done in duplicate with triplicate sets of readings for each assay point. Only T(4) prepared with 5% dextrose water (D5W) containing 1 mg/mL albumin remained constant, stable, predictable and accurate over time under various conditions. Other methods of preparation lost drug by adhering to the plastic containers and tubing by as much as 40% of starting concentration. T(3) recovery in the presence of 1 mg/mL of albumin was 107±2% (mean±standard error of the mean) of anticipated drug concentrations. We conclude from this series of experiments that to maintain an accurate and stable dosing of patients receiving intravenous thyroid hormones, 1 mg/mL of albumin must be added to the infusate to prevent lost on the plastic intravenous tubing.

Neuroprotection in a rabbit model of intraventricular haemorrhage by cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-alpha inhibition.

Intraventricular haemorrhage is a major complication of prematurity that results in neurological dysfunctions, including cerebral palsy and cognitive deficits. No therapeutic options are currently available to limit the catastrophic brain damage initiated by the development of intraventricular haemorrhage. As intraventricular haemorrhage leads to an inflammatory response, we asked whether cyclooxygenase-2, its derivative prostaglandin E2, prostanoid receptors and pro-inflammatory cytokines were elevated in intraventricular haemorrhage; whether their suppression would confer neuroprotection; and determined how cyclooxygenase-2 and cytokines were mechanistically-linked. To this end, we used our rabbit model of intraventricular haemorrhage where premature pups, delivered by Caesarian section, were treated with intraperitoneal glycerol at 2 h of age to induce haemorrhage. Intraventricular haemorrhage was diagnosed by head ultrasound at 6 h of age. The pups with intraventricular haemorrhage were treated with inhibitors of cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α; and cell-infiltration, cell-death and gliosis were compared between treated-pups and vehicle-treated controls during the first 3 days of life. Neurobehavioural performance, myelination and gliosis were assessed in pups treated with cyclooxygenase-2 inhibitor compared to controls at Day 14. We found that both protein and messenger RNA expression of cyclooxygenase-2, prostaglandin E2, prostanoid receptor-1, tumour necrosis factor-α and interleukin-1ß were consistently higher in the forebrain of pups with intraventricular haemorrhage relative to pups without intraventricular haemorrhage. However, cyclooxygenase-1 and prostanoid receptor 2-4 levels were comparable in pups with and without intraventricular haemorrhage. Cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α inhibition reduced inflammatory cell infiltration, apoptosis, neuronal degeneration and gliosis around the ventricles of pups with intraventricular haemorrhage. Importantly, cyclooxygenase-2 inhibition alleviated neurological impairment, improved myelination and reduced gliosis at 2 weeks of age. Cyclooxygenase-2 or prostanoid receptor-1 inhibition reduced tumour necrosis factor-α level, but not interleukin-1ß. Conversely, tumour necrosis factor-α antagonism did not affect cyclooxygenase-2 expression. Hence, prostanoid receptor-1 and tumour necrosis factor-α are downstream to cyclooxygenase-2 in the inflammatory cascade induced by intraventricular haemorrhage, and cyclooxygenase-2-inhibition or suppression of downstream molecules--prostanoid receptor-1 or tumour necrosis factor-α--might be a viable neuroprotective strategy for minimizing brain damage in premature infants with intraventricular haemorrhage.

Delivery of gastroschisis patients before 37 weeks of gestation is associated with increased morbidities.

BACKGROUND: Despite advances in the care of neonates with gastroschisis, patients present with significant morbidities. Preterm delivery of neonates with gastroschisis is often advocated to avoid the intestinal damage that may be sustained with prolonged exposure to amniotic fluid. However, preterm delivery may impose additional morbidities to this disease process. METHODS: We conducted a retrospective review of patients with gastroschisis born from 1989 to 2007. Demographic and clinical data were collected. Preterm healthy neonates, with gestational age from 26 to 36 weeks, were used as controls. RESULTS: Preterm infants with gastroschisis had a 14 times higher risk for any of the recorded morbidities. As compared to term neonates with gastroschisis, preterm neonates with gastroschisis had a higher rate of sepsis, longer duration to reach full enteral feedings, and longer length of stay. Although the preterm infants with gastroschisis were less likely to be small for gestational age at birth, they were as likely as the term infants with gastroschisis to have failure to thrive at discharge and had a greater drop in weight percentile during hospitalization. CONCLUSIONS: Preterm delivery should be avoided because there is no clear benefit to the gut in avoiding derivative injuries. Meticulous attention should be given to the nutritional needs of patients with gastroschisis.

Oxidative-nitrosative stress in a rabbit pup model of germinal matrix hemorrhage: role of NAD(P)H oxidase.

BACKGROUND AND PURPOSE: Germinal matrix hemorrhage-intraventricular hemorrhage is the most common neurological problem of premature infants. Despite this, mechanisms of brain injury from intraventricular hemorrhage are elusive. We hypothesized that germinal matrix hemorrhage-intraventricular hemorrhage, by induction of NAD(P)H oxidases, might cause oxidative/nitrosative stress contributing to brain injuries and that NAD(P)H oxidase inhibition could offer neuroprotection. METHODS: To test this hypothesis, we exploited our rabbit pup model of glycerol-induced germinal matrix hemorrhage-intraventricular hemorrhage. We delivered rabbit pups prematurely (E29) by cesarean section and administered intraperitoneal glycerol at 2 hours postnatal age. Free-radical adducts, including nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine as well as O(2)(.-) and H(2)O(2) levels were measured in the forebrain. To determine the source of free-radical generation, we used inhibitors for NAD(P)H oxidase (apocynin), xanthine oxidase (allopurinol), cyclo-oxygenase-2 (indomethacin), or nitric oxide synthases (L-NAME). Intraventricular hemorrhage pups were treated with apocynin and cell death was compared between apocynin-treated and vehicle-treated pups. RESULTS: Nitrotyrosine, 4-hyroxynonenal, and 8-hydroxy-deoxyguanosine levels were higher in pups with intraventricular hemorrhage than controls. Likewise, O(2)(.-) and H(2)O(2) levels were significantly greater in both the periventricular area and cerebral cortex of pups with intraventricular hemorrhage than controls. In pups with intraventricular hemorrhage, reactive oxygen species production was more in the periventricular area than in the cortex. Apocynin, but not allopurinol, indomethacin, or nitric oxide synthases, inhibited reactive oxygen species generation. Importantly, apocynin reduced cell death in pups with intraventricular hemorrhage. CONCLUSIONS: Activation of NAD(P)H oxidase was the predominant mechanism of free-radical generation in pups with intraventricular hemorrhage. NAD(P)H oxidase inhibition by apocynin might suppress reactive oxygen species production and confer neuroprotection in premature infants with intraventricular hemorrhage.

Outbreak of Acinetobacter infection in extremely low birth weight neonates.

OBJECTIVE: We describe the first outbreak of multiple drug-resistant Acinetobacter baumannii (MDR-Ab) in a neonatal intensive care unit in the United States. DESIGN/METHODS: MDR-Ab was identified in the blood of a 24-week gestation, 7-day-old extremely low birth weight neonate. Multiple samplings of surveillance surface cultures were performed on exposed and nonexposed neonates. Enhanced infection control measures were implemented. Pulsed-field gel electrophoresis was performed to determine the genetic relatedness of the MDR-Ab isolates. Medical records were reviewed for all exposed patients. RESULTS: MDR-Ab was recovered from 6 additional neonates. Of these 7 MDR-Ab (index + 6) neonates, 4 died, 3 of whom had positive blood cultures. All affected neonates were born between 23 to 26 weeks gestational age, and were <7 days postnatal age and <750 g (430-720) at the time of exposure. All were housed within the same room as the index case. None of the other 5 exposed neonates older than postnatal day 7 or weighing >750 g at birth were affected. No additional cases occurred outside the original room. Pulsed-field gel electrophoresis was consistent with a clonal origin, identical to MDR-Ab recovered from the referring hospital. CONCLUSIONS: This MDR-Ab outbreak was rapidly controlled with enhanced infection control measures and was novel in that it affected only <750 g neonates, at < or =26 weeks gestational age, and < or =7 days postnatal age at the time of exposure, suggesting that invasive Ab has a special affinity for damaged or nonkeratinized immature skin in developmentally immature immunologic hosts.

Outbreak of parainfluenza virus type 3 in a neonatal intensive care unit.

Parainfluenza virus (PIV) causes > 30% of all acute respiratory infections in infants and children and is second only to respiratory syncytial virus as a cause of lower respiratory tract infection. However in the neonatal intensive care unit (NICU), PIV outbreaks are highly uncommon. This case report describes an outbreak of 3 cases of PIV type 3 in a regional NICU.