Janine Y. Khan

Developmental changes in murine brain antioxidant enzymes.

Reactive oxygen species produced in cells during normal aerobic metabolism have the ability to induce lipid peroxidation and protein oxidation; therefore, their detoxification and elimination are necessary for physiologic cellular activity and survival. The changes in neuronal antioxidant enzymes from fetal life to adulthood have not been fully described. We investigated protein expression, using Western blot analysis, and enzymatic activity of the antioxidant system—copper-zinc superoxide dismutase (SOD), manganese SOD, catalase, and glutathione peroxidase, as well as reduced glutathione level as an indicator of the nonenzymatic system—in CD1 murine brain at embryonic d 18 (E18), and postnatal d 1 (P1), d 4, d 7, d 14, and d 21. Copper-zinc SOD and glutathione peroxidase protein levels were low, whereas manganese SOD and catalase protein levels were high at E18 and P1. Total SOD activity was high at E18 and P1 and paralleled elevated manganese SOD activity; however, copperzinc SOD activity was relatively unchanged throughout development. Catalase activity doubled and glutathione peroxidase activity tripled between E18 and P1. Reduced glutathione increased between E18 and P1. Except for catalase and manganese SOD, peak protein levels do not occur until later developmental ages. We suggest that as the fetus moves from an in utero hypoxic to a relatively hyperoxic environment with an approximate 4-fold elevation in oxygen concentration, these developmental changes in antioxidant enzymes are compensatory mechanisms aimed at protecting the newborn from oxidative stress. These data will be important in our future understanding of the mechanisms by which hypoxia mediates injury in the immature and the mature brain.

Beyond Necrotizing Enterocolitis Prevention: Improving Outcomes with an Exclusive Human Milk–Based Diet

Abstract Objective: The aim of this study was to compare outcomes of infants pre and post initiation of a feeding protocol providing an exclusive human milk–based diet (HUM). Materials and Methods: In a multicenter retrospective cohort study, infants with a birth weight <1,250 g who received a bovine-based diet (BOV) of mothers own milk fortified with bovine fortifier and/or preterm formula were compared to infants who received a newly introduced HUM feeding protocol. Infants were excluded if they had major congenital anomalies or died in the first 12 hours of life. Data were collected 2–3 years prior to and after introduction of an exclusive HUM diet. Primary outcomes were necrotizing enterocolitis (NEC) and mortality. Secondary outcomes included late-onset sepsis, retinopathy of prematurity (ROP), and bronchopulmonary dysplasia (BPD). Results: A total of 1,587 infants were included from four centers in Texas, Illinois, Florida, and California. There were no differences in baseline demographics or growth of infants. The HUM group had significantly lower incidence of proven NEC (16.7% versus 6.9%, p < 0.00001), mortality (17.2% versus 13.6%, p = 0.04), late-onset sepsis (30.3% versus 19.0%, p < 0.00001), ROP (9% versus 5.2%, p = 0.003), and BPD (56.3% versus 47.7%, p = 0.0015) compared with the BOV group. Conclusions: Extremely premature infants who received an exclusive HUM diet had a significantly lower incidence of NEC and mortality. The HUM group also had a reduction in late-onset sepsis, BPD, and ROP. This multicenter study further emphasizes the many benefits of an exclusive HUM diet, and demonstrates multiple improved outcomes after implementation of such a feeding protocol.

Developmental regulation of genes mediating murine brain glucose uptake

We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission.We examined the molecular mechanisms that mediate the developmental increase in murine whole brain 2-deoxyglucose uptake. Northern and Western blot analyses revealed an age-dependent increase in brain GLUT-1 (endothelial cell and glial) and GLUT-3 (neuronal) membrane-spanning facilitative glucose transporter mRNA and protein concentrations. Nuclear run-on experiments revealed that these developmental changes in GLUT-1 and -3 were regulated posttranscriptionally. In contrast, the mRNA and protein levels of the mitochondrially bound glucose phosphorylating hexokinase I enzyme were unaltered. However, hexokinase I enzyme activity increased in an age-dependent manner suggestive of a posttranslational modification that is necessary for enzymatic activation. Together, the postnatal increase in GLUT-1 and -3 concentrations and hexokinase I enzymatic activity led to a parallel increase in murine brain 2-deoxyglucose uptake. Whereas the molecular mechanisms regulating the increase in the three different gene products may vary, the age-dependent increase of all three constituents appears essential for meeting the increasing demand of the maturing brain to fuel the processes of cellular growth, differentiation, and neurotransmission.

Growth and Development in Extremely Low Birth Weight Infants After the Introduction of Exclusive Human Milk Feedings

Objective To estimate associations of exclusive human milk (EHM) feedings with growth and neurodevelopment through 18 months corrected age (CA) in extremely low birth weight (ELBW) infants. Study Design ELBW infants admitted from July 2011 to June 2013 who survived were reviewed. Infants managed from July 2011 to June 2012 were fed with bovine milk‐based fortifiers and formula (BOV). Beginning in July 2012, initial feedings used a human milk‐based fortifier to provide EHM feedings. Infants were grouped on the basis of feeding regimen. Primary outcomes were the Bayley‐III cognitive scores at 6, 12, and 18 months and growth. Results Infants (n = 85; 46% received EHM) were born at 26 ± 1.9 weeks (p = 0.92 between groups) weighing 776 ± 139 g (p = 0.67 between groups). Cognitive domain scores were similar at 6 months (BOV: 96 ± 7; EHM: 95 ± 14; p = 0.70), 12 months (BOV: 97 ± 10; EHM: 98 ± 9; p = 0.86), and 18 months (BOV: 97 ± 16; EHM: 98 ± 14; p = 0.71) CA. Growth velocity prior to discharge (BOV: 12.1 ± 5.2 g/kg/day; EHM: 13.1 ± 4.0 g/kg/day; p = 0.33) and subsequent growth was similar between groups. Conclusion EHM feedings appear to support similar growth and neurodevelopment in ELBW infants as compared with feedings containing primarily bovine milk‐based products.

Effect of primary congenital hypothyroidism upon expression of genes mediating murine brain glucose uptake.

Using hyt/hyt mice that exhibit naturally occurring primary hypothyroidism (n = 72) and Balb/c controls (n = 66), we examined the mRNA, protein, and activity of brain glucose transporters (Glut 1 and Glut 3) and hexokinase I enzyme at various postnatal ages (d 1, 7, 14, 21, 35, and 60). The hyt/hyt mice showed an age-dependent decline in body weight (p < 0.04) and an increase in serum TSH levels (p < 0.001) at all ages. An age-dependent translational/posttranslational 40% decline in Glut 1 (p = 0.02) with no change in Glut 3 levels was observed. These changes were predominant during the immediate neonatal period (d 1). A posttranslational 70% increase in hexokinase enzyme activity was noted at d 1 alone (p < 0.05) with no concomitant change in brain 2-deoxy-glucose uptake. This was despite a decline in the hyt/hyt glucose production rate. We conclude that primary hypothyroidism causes a decline in brain Glut 1 associated with no change in Glut 3 levels and a compensatory increase in hexokinase enzyme activity. These changes are pronounced only during the immediate neonatal period and disappear in the postweaned stages of development. These hypothyroid-induced compensatory changes in gene products mediating glucose transport and phosphorylation ensure an adequate supply of glucose to the developing brain during transition from fetal to neonatal life.

Clinical Outcomes Associated with a Failed Infant Car Seat Challenge

Objective To assess comorbid conditions and clinical outcomes among late preterm and low birth weight term infants (<2.5 kg) who failed the Infant Car Seat Challenge (ICSC) on the Mother‐Baby Unit. Study design This was a retrospective chart review of consecutive infants who failed ICSC on the Mother‐Baby Unit and were subsequently admitted to the neonatal intensive care unit at Prentice Womens Hospital between January 1, 2009, and December 31, 2015. Regression models were used to estimate risk differences (RDs) with 95% CIs for factors related to length of stay. Results A total of 148 infants were studied (43% male; 37% delivered via cesarean). ICSC failure in the Mother‐Baby Unit was due to desaturation, bradycardia, and tachypnea in 59%, 37%, and 4% of infants, respectively. During monitoring on the neonatal intensive care unit, 39% of infants experienced apnea (48% in preterm vs 17% in term infants) in the supine position, 19% received phototherapy, and 2% and 6.8% received nasogastric and thermoregulatory support, respectively. Univariate predictors of increased duration of stay (days) were younger gestational age, apnea, nasogastric support, intravenous fluids, and antibiotics (all P < .05). In multivariable analysis adjusted for gestational age and discharge weight, only apnea (RD, 4.87; 95% CI, 2.99‐6.74; P < .001), administration of antibiotics (RD, 3.25; 95% CI, 0.29‐6.21; P < .032), and intravenous fluid support (RD, 4.87; 95% CI, 0.076‐9.66; P < .047) remained independent predictors of a longer duration of stay. Conclusion Infants who failed ICSC were at risk for comorbid conditions that prolonged hospital stay beyond the neonatal intensive care unit observation period. Almost one‐half of late preterm infants who failed ICSC had apnea events in the supine position.

Evaluation of Introduction of a Delayed Cord Clamping Protocol for Premature Neonates in a High-volume Maternity Center

OBJECTIVE To evaluate adherence to a delayed cord clamping protocol for preterm births in the first 2 years after its introduction, perform a quality improvement assessment, and determine neonatal outcomes associated with protocol implementation and adherence. METHODS This is a retrospective cohort study of women delivering singleton neonates at 23-32 weeks of gestation in the 2 years before (preprotocol) and 2 years after (postprotocol) introduction of a 30-second delayed cord clamping protocol at a large-volume academic center. This policy was communicated to obstetric and pediatric health care providers and nurses and reinforced with intermittent educational reviews. Barriers to receiving delayed cord clamping were assessed using χ tests and multivariable logistic regression. Neonatal outcomes then were compared between all neonates in the preprotocol period and all neonates in the postprotocol period and between all neonates in the preprotocol period and neonates receiving delayed cord clamping in the postprotocol period using multivariable linear and logistic regression analyses. RESULTS Of the 427 eligible neonates, 187 were born postprotocol. Of these, 53.5% (n=100) neonates received delayed cord clamping according to the protocol. The rate of delayed cord clamping preprotocol was 0%. Protocol uptake and frequency of delayed cord clamping increased over the 2 years after its introduction. In the postprotocol period, cesarean delivery was the only factor independently associated with failing to receive delayed cord clamping (adjusted odds ratio [OR] 0.49, 95% confidence interval [CI] 0.25-0.96). In comparison with the preprotocol period, those who received delayed cord clamping in the postprotocol period had significantly higher birth hematocrit (β=2.46, P=.007) and fewer blood transfusions in the first week of life (adjusted OR 0.49, 95% CI 0.25-0.96). CONCLUSION After introduction of an institutional delayed cord clamping protocol followed by continued health care provider education and quality feedback, the frequency of delayed cord clamping progressively increased. Compared with historical controls, performing delayed cord clamping in eligible preterm neonates was associated with improved neonatal hematologic indices, demonstrating the effectiveness of delayed cord clamping in a large-volume maternity unit.

City-wide Surveillance for the Occurrence of Respiratory Syncytial Virus(RSV) Respiratory Tract Infections in a Population of High Risk Infants ≤32 weeks Gestational Age † 1453

RSV IGIV has been recommended for use in high-risk infants ≤35 wks GA. In 1996, the following criteria were adopted for RSV IGIV use in all Level III NICUs in the City of Pittsburgh: 1) <1yr of age with chronic lung disease(CLD) - mechanical ventilation, supplemental oxygen, steroids and/or diuretics beyond 14 days of life, 2) ≤32 wks GA and <6 months chronologic age with high risk of non-preventable exposure to RSV (sibs in preschool or daycare). The purpose of this study was to determine the prevalence of RSV infection and the effectiveness of RSV IGIV in reducing RSV-associated hospitalization in a city-wide population of infants ≤32 wks GA. All infants born at ≤32 wks GA from 7/1/96-4/30/97 who met criteria were eligible to receive 1 to 6 doses of RSV IGIV (given monthly from 11/1/96-4/30/97). Parents were contacted at intervals through 6/30/97 to determine RSV IGIV treatment, occurrence of RSV infection and risk factors. We identified 282 infants ≤32 wks GA; 205 families were contacted (73%). Mean GA and birthweight were 29.5 wks ± 2.4 and 1338 g ± 402 respectively. Of the 53 infants eligible for RSV IGIV, 4 had no doses and 7 received IM RSV. Forty-two infants received 78% of eligible doses of RSV IGIV and one was hospitalized with RSV (2.4%). Twelve infants ineligible for RSV IGIV were hospitalized for RSV infection (8.1%). Of these 13 hospitalized infants, 9 had no recognized risk factors except prematurity, 1 had CLD (RSV-eligible, received 4 of 6 doses), 1 attended daycare and 2 had sibs in daycare or preschool. Mean age at hospitalization was 15 weeks ± 11 and mean length of stay was 8 days ± 8. Ten infants developed an oxygen requirement, 1 was ventilated. There were no infant deaths. We conclude that with 92% compliance with RSV IGIV administration in Pittsburgh using the above criteria, there was a decrease in RSV-associated hospitalization in at-risk infants ≤32 weeks with a case reduction rate of 70%. This study may indicate that current RSV IGIV administration criteria for treatment of high-risk infants ≤35 wks GA may be excessive. Cost analysis will be performed to determine the cost effectiveness of this strategy.

Primary hypothyroidism alters expression and function of the hexokinase I gene which mediates brain glucose phosphorylation. † 1390

Primary hypothyroidism alters expression and function of the hexokinase I gene which mediates brain glucose phosphorylation. † 1390

Brain Hexokinase I Expression and Function during Development. † 1391

Glucose, an essential substrate for brain growth, cellular maturation, and oxidative metabolism is transported across the blood-brain barrier, into neurons and glial cells. Intracellularly glucose is phosphorylated into glucose-6-phosphate by the hexokinase I (hxl) enzyme. In the adult brain, glucose phosphorylation comprises the rate-limiting step in the process of glucose uptake. To determine the ontogeny of this critical rate limiting step, we examined the brain hxl expression (Northern blots), concentration (Western blots), enzymatic activity (NADPNADPH conversion by spectrophotometric assay) and function (3H-2-deoxy-glucose uptake) in Balb-C mice at 1d(n=6), 14d (n=6), and 35d (n=6) postnatal ages. Hxl mRNA and protein concentrations declined 20-50% (p < 0.05) while activity increased 2-fold between the 1d and 14d or 35d brains. In contrast, a six-fold increase in3 H-2-deoxy-glucose uptake (p < 0.05) which quantitates glucose transport and phosphorylation was noted between the 1d and 14d old mice. Our previous investigation demonstrated a 3-fold increase in brain glucose transporter expression and levels (particularly neuronal Glut 3) between the 1d and 14d mouse brains (Ped Res 39:91A, 1996). We conclude that 1] brain Hxl enzyme activity and function peak by a post-translational mechanism at the 14d postnatal age, 2] the age-related increase in brain Hxl enzyme activity along with the previous observation of a parallel increase in brain glucose transporter concentrations substantiate the age-related increase in brain 2-deoxyglucose uptake with a peak at 14d postnatal age. We speculate that this age-dependent increase in the mechanisms mediating brain glucose uptake 1] may be initiated by the physiological surge in thyroid hormone levels and activity that occurs at 14d of age, and 2] is critical for fueling the process of rapid brain growth and cellular development that occurs at 14d postnatal age in preparation for acquiring the specialized function of neurotransmission.