Screening Echocardiography Identifies Risk Factors for Pulmonary Hypertension at Discharge in Premature Infants with Bronchopulmonary Dysplasia.

HYPOTHESIS: Premature infants with bronchopulmonary dysplasia (BPD) are at increased risk of secondary pulmonary hypertension (BPD-PH). Prior studies yielded mixed results on the utility of echocardiographic screening at 36 weeks post-menstrual age (PMA). We present our experience using echocardiographic screening at the time of BPD diagnosis to identify infants at highest risk of BPD-PH at discharge. MATERIALS AND METHODS: Retrospective cohort analysis of clinical/ demographic data and screening echocardiograms in patients with BPD. Discharge echocardiograms identified infants with or without BPD-PH at discharge. 36 weeks PMA screening echocardiograms and clinical data were then reviewed to identify which factors were associated with increased odds of BPD-PH at discharge. Associations between echocardiographic findings were evaluated with 2- and 3-variable models to predict increased risk of BPD-PH at discharge. RESULTS: In our cohort of 64 infants with severe BPD, BPD-PH was present in 22/64 (34%) infants at discharge. There were no clinical differences at time of 36 weeks PMA screening evaluation (mean PMA 36.6 ± 2.9 weeks). PH at screening was poorly predictive of PH at discharge as PH at screening resolved in 49% of patients. However, having an ASD, RV dilation, hypertrophy, or reduced function on screening, especially in combination, were associated with BPD-PH at discharge. CONCLUSION: In our cohort of premature infants with BPD, 36 weeks PMA screening echocardiogram identified patients at increased risk for BPD-PH at discharge when ASD, RVH, or impaired RV function were present. Larger prospective studies are indicated to validate these findings.

Increased excitability and excitatory synaptic transmission during in vitro ischemia in the neonatal mouse hippocampus.

OBJECTIVE: The present study tested the hypothesis that exposure to in vitro hypoxia-ischemia alters membrane properties and excitability as well as excitatory synaptic transmission of CA1 pyramidal neurons in the neonatal mouse. METHODS: Experiments were conducted in hippocampal slices in P7-P9 C57Bl/6 mice using whole-cell patch clamp in current- and voltage-clamp mode. Passive membrane potential (Vm), input resistance (Rin) and active (action potential (AP) threshold and amplitude) membrane properties of CA1 pyramidal neurons were assessed at baseline, during 10 min in vitro ischemia (oxygen-glucose deprivation (OGD)) and during reoxygenation. Spontaneous and miniature excitatory post-synaptic currents (s and mEPSCs) were studied under similar conditions. RESULTS: OGD caused significant depolarization of CA1 pyramidal neurons as well as decrease in AP threshold and increase in AP amplitude. These changes were blocked by the application of tetrodotoxin (TTX), indicating Na(+) channels' involvement. Following 10 min of reoxygenation, significant membrane hyperpolarization was noted and it was associated with a decrease in Rin. AP threshold and amplitude returned to baseline during that stage. sEPSC and mEPSC frequency increased during both OGD and reoxygenation but their amplitude remained unchanged. Additionally, we found that OGD decreases Ih (hyperpolarization activated current) in CA1 neurons from neonatal mice and this effect persists during reoxygenation. SIGNIFICANCE: These results indicate that in vitro ischemia leads to changes in membrane excitability mediated by sodium and potassium channels. Further, it results in enhanced neurotransmitter release from presynaptic terminals. These changes are likely to represent one of the mechanisms of hypoxia/ischemia-mediated seizures in the neonatal period.

Salmonella berta meningitis in a term neonate.

We report the case of a 37-week male infant born via spontaneous vaginal delivery who developed Salmonella berta sepsis and meningitis. The infant was born to a mother with active diarrhea and stool cultures growing S. berta. On day 3, the infant developed poor feeding, lethargy, apnea and bradycardia prompting a sepsis evaluation. Blood, stool and cerebrospinal fluid cultures were positive for S. berta. An electroencephalogram performed for posturing revealed neonatal status epilepticus. Extensive bilateral periventricular venous hemorrhagic infarctions with multiple herniations were seen on brain magnetic resonance imaging. The infant's condition continued to deteriorate despite maximal support and care was redirected towards comfort measures.

Implementation of a 'Hypothermia for HIE' program: 2-year experience in a single NICU.

Hypothermia has been shown to be neuroprotective in some newborns with moderate-to-severe perinatal hypoxic-ischemic encephalopathy (HIE). In 2006, the American Academy of Pediatrics recommended that institutions that choose to use therapeutic hypothermia do so in the context of a rigorous protocol, with systematic collection of patient data including neurodevelopmental follow-up. In this report, we describe our experience with implementation of a 'Hypothermia for HIE' program in a single tertiary care Neonatal Intensive Care Unit (NICU). Important components of the program include detailed protocols, staff and outreach education, early initiation of cooling in both inborn and outborn patients, maintaining stable hypothermia during neonatal transport, and comprehensive neurologic evaluation including serial EEGs, brain MRI and neurodevelopmental follow-up. In the first 2 years of the program, we have used hypothermia therapy in 21 patients, 18 with perinatal and 3 with early postnatal events leading to HIE. Eleven of fifteen outborn patients were cooled prior to and during transport, resulting in initiation of therapy 3 h sooner than if therapy had been delayed until arrival at our center. While lowering the body temperature of encephalopathic newborns is not difficult, addressing the complex medical problems of this vulnerable group of patients requires an experienced multidisciplinary team in regional referral centers.

Hypoxia modifies nuclear calcium uptake pathways in the cerebral cortex of the guinea-pig fetus.

Nuclear Ca2+ signals are thought to play a critical role in the initiation and progression of programmed cell death. The present study tests the hypothesis that hypoxia alters nuclear Ca2+ transport pathways and leads to an increase in nuclear Ca(2+)-influx in cerebral cortical neuronal nuclei. To test this hypothesis the effect of tissue hypoxia on high affinity Ca(2+)-ATPase activity and the binding characteristics of inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) receptors were studied in neuronal nuclei from the cerebral cortex of guinea-pig fetuses. Results show increased high-affinity Ca(2+)-ATPase activity (nmol/mg protein/h) in the hypoxic group 969.7+/-79 as compared with 602.4+/-90.9 in the normoxic group, P<0.05. The number of IP3 receptors (Bmax, fmol/mg protein) increased from 61+/-21 in the normoxic group to 164+/-49 in the hypoxic group, P<0.05. K(d) values did not change following hypoxia. In contrast, IP4 receptor Bmax (fmol/mg protein) and K(d) (nM) values increased from 360+/-32 in the normoxic group to 626+/-136 in the hypoxic group (P<0.001) and, from 26+/-1 in the normoxic group to 61+/-9 in the hypoxic group (P<0.001), respectively. 45Ca(2+)-influx (pmol/mg protein) significantly increased from 6.3+/-1.9 in the normoxic group to 10.9+/-1.1 the hypoxic group (P<0.001). The data show that hypoxia modifies nuclear Ca2+ transport pathways and results in increased nuclear Ca(2+)-influx. We speculate that hypoxia increases nuclear Ca2+ uptake from the cytoplasm to the nucleoplasm, resulting in increased transcription of proapoptotic genes and subsequent activation of programmed cell death pathways.

Nitration is a mechanism of regulation of the NMDA receptor function during hypoxia.

The present study tested the hypothesis that nitration is a mechanism of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor. To test this hypothesis the effect of hypoxia on the nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor was determined. Furthermore, the effect of administration of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNLA) on the hypoxia-induced nitration of the NMDA receptor subunits as well as the NMDA receptor-mediated Ca2+ influx, an index of NMDA receptor-ion channel function, were determined in cortical synaptosomes. Studies were performed in newborn piglets divided into normoxic, hypoxic and hypoxic-NNLA groups. Hypoxia was induced by decreasing the FiO(2) to 0.07-0.09 for 60 min. Cerebral tissue hypoxia was confirmed by determining the levels of high energy phosphates ATP and phosphocreatine. Nitration of the NMDA receptor subunits was determined by immunoprecipitation using specific antibodies and western blot analysis. NMDA receptor-ion channel-mediated Ca2+ influx was determined using 45Ca2+. There was a significant increase in the nitrated NR1, NR2A and NR2B subunits following hypoxia: 104+/-11 vs. 275+/-18 optical density (OD)xmm(2) for NR1 (P<0.05), 212+/-36 vs. 421+/-16 ODxmm(2) for NR2A (P<0.05) and 246+/-44 vs. 360+/-26 ODxmm(2) for NR2B (P<0.05). This increase in nitrated NR1, NR2A and NR2B subunits of the NMDA receptor was prevented by the administration of NNLA prior to hypoxia (NR1 160+/-19, P=NS, NNLA vs. normoxic; NR2A 304+/-49, P=NS, NNLA vs. normoxic, and NR2B 274+/-19, P=NS, NNLA vs. normoxic). The increase in nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increased as a function of decreased cerebral high-energy phosphates, ATP and phosphocreatine, during hypoxia. Furthermore, NOS blockade prior to hypoxia resulted in prevention of the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. Our results demonstrate that hypoxia results in increased nitration of the NMDA receptor subunits and that administration of an NOS inhibitor prior to hypoxia prevents the hypoxia-induced nitration of the NMDA receptor subunits as well as the hypoxia-induced increase in NMDA receptor-mediated Ca2+ influx. We conclude that nitration is a mechanism of modification of the NMDA receptor function during hypoxia in the newborn piglet brain.

Acute hypoxia-induced alterations of calbindin-D28k immunoreactivity in cerebellar Purkinje cells of the guinea pig fetus at term.

Purkinje cells (PCs) are vulnerable to hypoxic/ischemic insults and rich in calcium and calcium-buffering/sequestering systems, including calcium-binding proteins (CaBPs). Calbindin-D28k is an EF-hand CaBP, which is highly expressed in PCs where it acts primarily as a cellular Ca++ buffer. Elevation of [Ca++] in the cytosol and nuclei of PCs is pivotal in hypoxic/ischemic cell death. We hypothesize that hypoxia results in decreased concentration, or availability of calbindin-D28k in PCs, thereby decreasing their buffering capacity and resulting in increase of intracellular and intranuclear [Ca++]. Cerebellar tissues from normoxic fetuses were compared to fetuses obtained from term pregnant guinea pigs exposed to hypoxia [7% FiO2] for 60 min. The pregnant guinea pigs were either killed upon delivery immediately following hypoxia (Hx0h) or were subsequently allowed to recover for 24 h (Hx24h) or 72 h (Hx72h). Fetal brain hypoxia was documented biochemically by a decrease in brain tissue levels of ATP and phosphocreatine. Compared to normoxic fetuses, there is a predominantly somatodendritic loss or decrease of calbindin-D28k immunohistochemical staining in PCs of Hx0h (p < 0.005), Hx24h (p < 0.05), and Hx72h (p < 0.005) fetuses. Hypoxia-induced alterations of calbindin-D28k immunoreactivity are qualitatively similar at all time points and include a distinctive intranuclear localization in subpopulations of PCs. A similar trend is demonstrated by immunoblotting. Subpopulations of TUNEL+/calbindin-D28k- PCs lacking morphologic features of apoptosis or necrosis are demonstrated in Hx24h and Hx72h fetuses. The present study demonstrates an abrogating effect of perinatal hypoxia on calbindin-D28k immunoreactivity in cerebellar PCs. The perturbation of this Ca++ buffer protein in hypoxia-induced neuronal injury may herald delayed cell death or degeneration.

Phosphorylation of Bcl-2 and Bax proteins during hypoxia in newborn piglets.

Studies indicate that phosphorylated Bcl-2 cannot form a heterodimer with Bax and thus may lose its antiapoptotic potential. The present study tests the hypothesis that graded hypoxia in cerebral tissue induces the phosphorylation of Bcl-2, thus altering the heterodimerization of Bcl-2 with Bax and subsequently leading to apoptosis. Anesthetized, ventilated newborn piglets were assigned to a normoxic and a graded hypoxic group. Cerebral cortical neuronal nuclei were isolated and immunoprecipitated; immune complexes were separated and reacted with Bcl-2 and Bax specific antibodies. The results show an increased level of serine/tyrosine phosphorylated Bcl-2 in nuclear membranes of hypoxic animals. The level of phosphorylated Bcl-2 protein increased linearly with decrease in tissue PCr. The level of phosphorylated Bax in the neuronal nuclear membranes was independent of cerebral tissue PCr. The data shows that during hypoxia, there is increased phosphorylation of Bcl-2, which may prevent its heterodimerization with Bax and lead to increased proapoptotic activity due to excess Bax in the hypoxic brain. Further increased phosphorylation of Bcl-2 may alter the Bcl-2/Bax-dependent antioxidant, lipid peroxidation and pore forming activity, as well as the regulation of intranuclear Ca2+ and caspase activation pathways. We speculate that increased phosphorylation of Bcl-2 in neuronal nuclear membranes is a potential mechanism of programmed cell death activation in the hypoxic brain.

Effect of graded hypoxia on cerebral cortical genomic DNA fragmentation in newborn piglets.

Previous studies have shown that hypoxia is associated with modification of the cerebral cortical nuclear membrane, leading to increased intranuclear calcium. The increased intranuclear calcium activates calcium-dependent endonucleases, resulting in DNA fragmentation. The present study tests the hypothesis that the fragmentation of neuronal genomic DNA increases with an increase in the degree of cerebral tissue hypoxia. Sixteen newborn piglets were anesthetized, ventilated and divided into normoxic and hypoxic groups with varying degrees of hypoxia. Cerebral hypoxia was documented biochemically by measuring tissue levels of ATP and phosphocreatine. Isolation of cerebral cortical neuronal nuclei and DNA and their purity was confirmed by standard techniques. DNA samples were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. In the hypoxic samples, multiple low-molecular-weight DNA fragments were present as a smear pattern from 200 to 2,000 base pairs. Levels of high-energy phosphates were compared to the area of each smear for each animal to correlate the degree of hypoxia with the degree of DNA fragmentation. DNA fragmentation increased when high-energy phosphate levels decreased. We conclude that there is a critical threshold value of oxidative metabolism beyond which there are progressive changes in the cortical neuronal cells, leading to DNA fragmentation.

Peroxynitrite-induced modification of the N-methyl-D-aspartate receptor in the cerebral cortex of the guinea pig fetus at term.

The present study tests the hypothesis that nitration is a potential mechanism of N-methyl-D-aspartate (NMDA) receptor modification, by assessing the effect of peroxynitrite in vitro on the glutamate and ion-channel sites of the NMDA receptor in the fetal guinea pig. Nitration of NMDA receptor subunits was confirmed by Western blot. Following peroxynitrite exposure, (3)H-MK-801 bindings show an increase in the B(max) and a decrease in the K(d), while (3)H-glutamate bindings show a decrease in the K(d) with no change in the B(max). We conclude that peroxynitrite regulates the NMDA receptor function by increasing the affinity of the ion-channel and glutamate sites, and by exposing additional ion-channel sites. We propose that nitration of the NMDA receptor is a potential mechanism for the regulation of the receptor during hypoxia.

Effect of nitric oxide synthase inhibition on high affinity Ca(2+)-ATPase during hypoxia in cerebral cortical neuronal nuclei of newborn piglets.

Previous studies have shown that during hypoxia, neuronal nuclear high affinity Ca(2+)-ATPase activity is increased in the cerebral cortex of newborn piglets. The present study tests the hypothesis that pretreatment with N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increase in high affinity Ca(2+)-ATPase activity in cortical neuronal nuclear membrane of newborn piglets. We also tested the hypothesis that nitration is a mechanism of elevation of the high affinity Ca(2+)-ATPase activity during hypoxia. Studies were performed in five normoxic, five hypoxic, and six NNLA-pretreated (40 mg/kg) hypoxic newborn piglets. Cerebral cortical neuronal nuclei were isolated and the high affinity Ca(2+)-ATPase activity was determined. Further, normoxic samples were aliquoted into two sub-groups for in vitro nitration with 0.5 mM peroxynitrite and subsequent determination of the high affinity Ca(2+)-ATPase activity. The activity increased from 309+/-40 nmol Pi/mg protein/h in the normoxic group to 520+/-108 nmol Pi/mg protein/h in the hypoxic group (P<0.05). In the NNLA-pretreated group, the activity was 442+/-53 nmol Pi/mg protein/h (P<0.05), which is 25% lower than in the hypoxic group. In the nitrated group the enzyme activity increased to 554+/-59 nmol Pi/mg protein/h (P<0. 05). Thus peroxynitrite-induced nitration in vitro increased the high affinity Ca(2+)-ATPase activity and NNLA administration in vivo partially prevented the hypoxia-induced increase in neuronal nuclear high affinity Ca(2+)-ATPase activity. We conclude that the hypoxia-induced increase in nuclear membrane high affinity Ca(2+)-ATPase activity is NO-mediated and that nitration of the enzyme is a mechanism of its modification.

NMDA receptor-mediated calcium influx in cerebral cortical synaptosomes of the hypoxic guinea pig fetus.

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca2+]i observed under hypoxic conditions is the result of an NMDA-mediated Ca2+ influx. Changes of [Ca2+]i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca2+]i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca2+]i was not observed in a Ca2+ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca2+]i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca2+ via the altered NMDA receptor with subsequent release of Ca2+ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets.

Bilirubin neurotoxicity can be mediated by numerous mechanisms due to its increased permeability in neuronal membranes. The present study tests the hypothesis that a prolonged bilirubin infusion modifies the N-methyl-D-aspartate (NMDA) receptor/ ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in seven control and six bilirubin-exposed piglets, 2-4 d of age. Piglets in the bilirubin group received a 35 mg/kg bolus of bilirubin followed by a 4-h infusion (25 mg/kg/h) of a buffer solution containing 0.1 N NaOH, 5% human albumin, and 0.055 Na2HPO4 with 3 mg/mL bilirubin. The final mean bilirubin concentration in the bilirubin group was 495.9 +/- 85.5 mumol/L (29.0 +/- 5.0 mg/dL). The control group received a bilirubin-free buffer solution. Sulfisoxazole was administered to animals in both groups. P2 membrane fractions were prepared from the cerebral cortex. [3H]MK-801 binding assays were performed to study NMDA receptor modification. The Bmax in the control and bilirubin groups were 1.20 +/- 0.10 (mean +/- SD) and 1.32 +/- 0.14 pmol/mg protein, respectively. The value for Kd in the control brains was 6.97 +/- 0.80 nM compared with 4.80 +/- 0.28 nM in the bilirubin-exposed brains (p < 0.001). [3H]Glutamate binding studies did not show a significant difference in the Bmax and Kd for the NMDA-specific glutamate site in the two groups. The results show that in vivo exposure to bilirubin increases the affinity of the receptor (decreased Kd) for [3H]MK-801, indicating that bilirubin modifies the function of the NMDA receptor/ion channel complex in the brain of the newborn piglet. We speculate that the affinity of bilirubin for neuronal membranes leads to bilirubin-mediated neurotoxicity, resulting in either short- or long-term disruption of neuronal function.