Focal Brain Injury Associated with a Model of Severe Hypoxic-Ischemic Encephalopathy in Nonhuman Primates.

Worldwide, hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal mortality and morbidity. To better understand the mechanisms contributing to brain injury and improve outcomes in neonates with HIE, better preclinical animal models that mimic the clinical situation following birth asphyxia in term newborns are needed. In an effort to achieve this goal, we modified our nonhuman primate model of HIE induced by in utero umbilical cord occlusion (UCO) to include postnatal hypoxic episodes, in order to simulate apneic events in human neonates with HIE. We describe a cohort of 4 near-term fetal Macaca nemestrina that underwent 18 min of in utero UCO, followed by cesarean section delivery, resuscitation, and subsequent postnatal mechanical ventilation, with exposure to intermittent daily hypoxia (3 min, 8% O2 3-8 times daily for 3 days). After delivery, all animals demonstrated severe metabolic acidosis (pH 7 ± 0.12; mean ± SD) and low APGAR scores (<5 at 10 min of age). Three of 4 animals had both electrographic and clinical seizures. Serial blood samples were collected and plasma metabolites were determined by 2-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS). The 4 UCO animals and a single nonasphyxiated animal (delivered by cesarean section but without exposure to UCO or prolonged sedation) underwent brain magnetic resonance imaging (MRI) on day 8 of life. Thalamic injury was present on MRI in 3 UCO animals, but not in the control animal. Following necropsy on day 8, brain histopathology revealed neuronal injury/loss and gliosis in portions of the ventrolateral thalamus in all 4 UCO, with 2 animals also demonstrating putamen/globus pallidus involvement. In addition, all 4 UCO animals demonstrated brain stem gliosis, with neuronal loss present in the midbrain, pons, and lateral medulla in 3 of 4 animals. Transmission electron microscopy imaging of the brain tissues was performed, which demonstrated ultrastructural white matter abnormalities, characterized by perinuclear vacuolation and axonal dilation, in 3 of 4 animals. Immunolabeling of Nogo-A, a negative regulator of neuronal growth, was not increased in the injured brains compared to 2 control animals. Using GC × GC-TOFMS, we identified metabolites previously recognized as potential biomarkers of perinatal asphyxia. The basal ganglia-thalamus-brain stem injury produced by UCO is consistent with the deep nuclear/brainstem injury pattern seen in human neonates after severe, abrupt hypoxic-ischemic insults. The UCO model permits timely detection of biomarkers associated with specific patterns of neonatal brain injury, and it may ultimately be useful for validating therapeutic strategies to treat neonatal HIE.

Serial plasma metabolites following hypoxic-ischemic encephalopathy in a nonhuman primate model.

Biomarkers that indicate the severity of hypoxic-ischemic brain injury and response to treatment and that predict neurodevelopmental outcomes are urgently needed to improve the care of affected neonates. We hypothesize that sequentially obtained plasma metabolomes will provide indicators of brain injury and repair, allowing for the prediction of neurodevelopmental outcomes. A total of 33 Macaca nemestrina underwent 0, 15 or 18 min of in utero umbilical cord occlusion (UCO) to induce hypoxic-ischemic encephalopathy and were then delivered by hysterotomy, resuscitated and stabilized. Serial blood samples were obtained at baseline (cord blood) and at 0.1, 24, 48, and 72 h of age. Treatment groups included nonasphyxiated controls (n = 7), untreated UCO (n = 11), UCO + hypothermia (HT; n = 6), and UCO + HT + erythropoietin (n = 9). Metabolites were extracted and analyzed using comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry and quantified by PARAFAC (parallel factor analysis). Using nontargeted discovery-based methods, we identified 63 metabolites as potential biomarkers. The changes in metabolite concentrations were characterized and compared between treatment groups. Further comparison determined that 8 metabolites (arachidonic acid, butanoic acid, citric acid, fumaric acid, lactate, malate, propanoic acid, and succinic acid) correlated with early and/or long-term neurodevelopmental outcomes. The combined outcomes of death or cerebral palsy correlated with citric acid, fumaric acid, lactate, and propanoic acid. This change in circulating metabolome after UCO may reflect cellular metabolism and biochemical changes in response to the severity of brain injury and have potential to predict neurodevelopmental outcomes.

Dexmedetomidine reduces cranial temperature in hypothermic neonatal rats.

BACKGROUND: The α2-adrenergic agonist dexmedetomidine (DEX) is increasingly used for prolonged sedation of critically ill neonates, but there are currently no data evaluating possible consequences of prolonged neonatal DEX exposure. We evaluated the pharmacokinetics and histological consequences of neonatal DEX exposure. METHODS: DEX was administered (s.c.) to naive (uninjured) neonatal Lewis rats to provide acute (25 µg/kg, ×1) or prolonged (25 µg/kg three times daily, ×2 or ×4 d) exposure. Therapeutic hypothermia was simulated using a water-cooled blanket. Cranial temperatures were measured using an infrared thermometer. DEX concentrations were measured by LC-MS in plasma and homogenized brainstem tissue for pharmacokinetic analysis. Cortex, cerebellum, and brainstem were evaluated for evidence of inflammation or injury. RESULTS: Prolonged neonatal DEX exposure was not associated with renal or brain pathology or indices of gliosis, macrophage activation, or apoptosis in either hypothermic or control rats. Plasma and brain DEX concentrations were tightly correlated. DEX peaked within 15 min in brain and reduced cranial temperature from 32 to 30 °C within 30 min after injection in cooled rats. CONCLUSION: Prolonged DEX treatment in neonatal rats was not associated with abnormal brain histology. These data provide reassuring preliminary results for using DEX with therapeutic hypothermia to treat near-term brain injury.

Characterization of health care provider attitudes toward parental involvement in neonatal resuscitation-related decision making in Mongolia.

The aim of this study was to characterize attitudes and practices among health care providers (HCPs) in Mongolia regarding parental involvement in neonatal resuscitation (NR)-related decisions. A voluntary, anonymous questionnaire was administered to 210 HCPs across 19 of 21 Mongolia provinces. Eligible HCPs included midwives, neonatologists, pediatricians, and obstetricians involved in neonatal-perinatal care in both rural and urban hospitals. A total of 210 pediatric HCPs were surveyed and 100 % completed all questions (response rate 100 %). Despite the absence of nation-wide guidelines, NR is uniformly performed at 32-weeks gestation across HCP professions and across rural/urban settings. Most HCPs (67 %) indicate that parents should be counseled about resuscitation, but only 9 % ask the parents if they want their extremely premature child resuscitated and only 17 % counsel the parents prior to birth of an at-risk infant. Most HCPs (72 %) prefer to unilaterally decide when to withdraw NR, and only 28 % indicated that both parents should be involved in the decision. Following a newborn's death, 75 % of all HCPs reported that they do explain the death to parents, although only 28 % reported receiving any training in parental grief counseling. For HCPs in Mongolia, a discrepancy exists between the perceived value of parental involvement and the actual practice of NR-related counseling. This report is a necessary first step toward understanding the factors that influence NR-related practices in Mongolia, and may serve as model for collecting these types of data in other low and middle income countries.

Concurrent erythropoietin and hypothermia treatment improve outcomes in a term nonhuman primate model of perinatal asphyxia.

BACKGROUND: Up to 65% of untreated infants suffering from moderate to severe hypoxic-ischemic encephalopathy (HIE) are at risk of death or major disability. Therapeutic hypothermia (HT) reduces this risk to approximately 50% (number needed to treat: 7-9). Erythropoietin (Epo) is a neuroprotective treatment that is promising as an adjunctive therapy to decrease HIE-induced injury because Epo decreases apoptosis, inflammation, and oxidative injury and promotes glial cell survival and angiogenesis. We hypothesized that HT and concurrent Epo will be safe and effective, improve survival, and reduce moderate-severe cerebral palsy (CP) in a term nonhuman primate model of perinatal asphyxia. METHODOLOGY: Thirty-five Macaca nemestrina were delivered after 15-18 min of umbilical cord occlusion (UCO) and randomized to saline (n = 14), HT only (n = 9), or HT+Epo (n = 12). There were 12 unasphyxiated controls. Epo (3,500 U/kg × 1 dose followed by 3 doses of 2,500 U/kg, or Epo 1,000 U/kg/day × 4 doses) was given on days 1, 2, 3, and 7. Timed blood samples were collected to measure plasma Epo concentrations. Animals underwent MRI/MRS and diffusion tensor imaging (DTI) at <72 h of age and again at 9 months. A battery of weekly developmental assessments was performed. RESULTS: UCO resulted in death or moderate-severe CP in 43% of saline-, 44% of HT-, and 0% of HT+Epo-treated animals. Compared to non-UCO control animals, UCO animals exhibit poor weight gain, behavioral impairment, poor cerebellar growth, and abnormal brain DTI. Compared to UCO saline, UCO HT+Epo improved motor and cognitive responses, cerebellar growth, and DTI measures and produced a death/disability relative risk reduction of 0.911 (95% CI -0.429 to 0.994), an absolute risk reduction of 0.395 (95% CI 0.072-0.635), and a number needed to treat of 2 (95% CI 2-14). The effects of HT+Epo on DTI included an improved mode of anisotropy, fractional anisotropy, relative anisotropy, and volume ratio as compared to UCO saline-treated infants. No adverse drug reactions were noted in animals receiving Epo, and there were no hematology, liver, or kidney laboratory effects. CONCLUSIONS/SIGNIFICANCE: HT+Epo treatment improved outcomes in nonhuman primates exposed to UCO. Adjunctive use of Epo combined with HT may improve the outcomes of term human infants with HIE, and clinical trials are warranted.

Perinatal asphyxia in a nonhuman primate model.

Perinatal asphyxia is a leading cause of brain injury in neonates, occurring in 2-4 per 1,000 live births, and there are limited treatment options. Because of their similarity to humans, nonhuman primates are ideal for performing preclinical tests of safety and efficacy for neurotherapeutic interventions. We previously developed a primate model of acute perinatal asphyxia using 12-15 min of umbilical cord occlusion. Continuing this research, we have increased cord occlusion time from 15 to 18 min and extended neurodevelopmental follow-up to 9 months. The purpose of this report is to evaluate the increase in morbidity associated with 18 min of asphyxia by comparing indices obtained from colony controls, nonasphyxiated controls and asphyxiated animals. Pigtail macaques were delivered by hysterotomy after 0, 15 or 18 min of cord occlusion, then resuscitated. Over the ensuing 9 months, for each biochemical and physiologic parameters, behavioral and developmental evaluations, and structural and spectroscopic MRI were recorded. At birth, all asphyxiated animals required resuscitation with positive pressure ventilation and exhibited biochemical and clinical characteristics diagnostic of hypoxic-ischemic encephalopathy, including metabolic acidosis and attenuated brain activity. Compared with controls, asphyxiated animals developed long-term physical and cognitive deficits. This preliminary report characterizes the acute and chronic consequences of perinatal asphyxia in a nonhuman primate model, and describes diagnostic imaging tools for quantifying correlates of neonatal brain injury as well as neurodevelopmental tests for evaluating early motor and cognitive outcomes.

Time course of C-reactive protein and inflammatory mediators after neonatal surgery.

OBJECTIVE: To characterize the perioperative course of C-reactive protein (CRP) and inflammatory mediators in neonates ≤44 weeks' corrected gestational age. STUDY DESIGN: Prospective study of CRP and inflammatory mediators interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor-α in 55 neonates undergoing thoracic or abdominal surgery. RESULTS: In the absence of infection, CRP increased after surgery, peaking on post-operative day 2. The perioperative patterns of CRP differed by diagnosis and inflammatory state. Surgery alone did not cause an increase in CRP because in 13 of 55 infants (24%), CRP remained <1.0 mg/dL at all time points. For thoracic procedures, patent ductus arteriosus ligation showed the least post-operative increase in CRP, and patients undergoing repair of congenital diaphragmatic hernia or tracheoesophageal fistula had a greater response. Abdominal procedures with low CRP response included repair of imperforate anus and pyloric stenosis, while gastroschisis repair and bowel reanastomosis after necrotizing enterocolitis were accompanied by a robust CRP response. IL-6 concentrations peaked on post-operative day 1 and correlated with the post-operative day 2 CRP peak (r=0.398, P=.004). The additional inflammatory mediators measured were not informative. CONCLUSIONS: The range and time course of perioperative CRP differ by diagnosis. Serial measurements may be more informative than CRP magnitude.

Erythropoietin for infants with hypoxic-ischemic encephalopathy.

PURPOSE OF REVIEW: Perinatal asphyxia, intraventricular hemorrhage and stroke are common causes of neonatal brain injury, with hypoxia-ischemia as the final common pathway of injury. Erythropoietin (Epo) has potential to lessen neurologic sequelae due to hypoxia-ischemia. The purpose of this review is to highlight new clinical trials and experimental evidence that expand our understanding of Epo as a potential treatment for perinatal brain injury. RECENT FINDINGS: Several trials of Epo treatment are reviewed: two phase I/II trials of high-dose Epo given to preterm infants established pharmacokinetic and safety profiles, and a trial of Epo treatment for term infants with moderate hypoxic-ischemic encephalopathy found reduced disability. Potential risks and benefits of high-dose Epo are discussed. New evidence related to Epo receptor expression, signal transduction pathways, and mechanisms of neuroprotection are reviewed. SUMMARY: Cautious optimism is warranted regarding the use of high-dose Epo as a treatment option for neonatal brain injury. To date, Epo has been well tolerated to use in neonatal populations and now studies of neuroprotective efficacy are underway.

Postnatal stress produces hyperglycemia in adult rats exposed to hypoxia-ischemia.

Fetal or early postnatal stressors may predispose infants to develop diabetes, metabolic syndrome, or stroke. We hypothesized that postnatal stress will predispose animals to develop metabolic syndrome and impair the physiologic response to hypoxic-ischemic brain injury. We characterized the short- and long-term physiologic responses to postnatal stress by examining corticosterone (CS), glucose metabolism, and brain injury in neonatal and adult rats exposed to hypoxia-ischemia (H-I). Rat pups were divided into three levels of postnatal stress from postnatal day (P) 3 to P7. All rats underwent unilateral brain injury on either P7 or P134. We measured brain injury, growth, blood pressure, urine/plasma CS, plasma leptin, insulin, and glucose before and after H-I. Postnatal stress increased neonatal CS production, exacerbated neonatal white matter injury, and was associated with adult hyperglycemia after H-I despite increased insulin production. There were no group differences in adult weight, blood pressure, or leptin. Postnatal stress exacerbated brain injury and produced adult hyperglycemia, triggered after hypoxia exposure, consistent with the hypotheses that neonates exposed to early stress are more vulnerable to hypoxia and may be predisposed to develop metabolic syndrome in adulthood. Prolonged maternal separation produced more hyperglycemia than did brief daily handling.

High-dose erythropoietin does not exacerbate retinopathy of prematurity in rats.

Preterm infants are at high risk of brain injury, and high-dose recombinant erythropoietin (rEpo) may be therapeutic. However, the effect of rEpo on the development of retinopathy of prematurity (ROP) is unknown. We hypothesized that (1) rEpo would cross the blood-eye barrier and (2) early rEpo would modulate ROP in a rat model. Epo concentrations were measured by ELISA from the plasma and the homogenized eye tissue at timed intervals after rEpo injection. Flat-mounted retinas were prepared from rats given rEpo (0, 5000, or 30,000 U/kg i.p. qid x 3) on postnatal d (P) 1-3 that were raised in room air (RA) or cyclic oxygen exposure (COE) with O2 cycling every 24 h between 50% and 10% for 14 d. Photomicrographs of the fluorescein- or ADPase-stained P20 retinas were examined. rEpo penetrated into the eye in a dose- and time-dependent manner. COE increased retinal vascular pathology and decreased vessel density compared with RA controls. The 30,000 U/kg dose of rEpo increased the ROP clock hour scores, but only in ADPase-stained tissues. In contrast, 5000 U/kg rEpo did not change the incidence or severity of ROP by any measure. High-dose rEpo may protect against preterm brain injury with minimal impact on ROP.

Microarray analysis of high-dose recombinant erythropoietin treatment of unilateral brain injury in neonatal mouse hippocampus.

Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of brain injury. Proposed mechanisms of neuroprotection include activation of gene pathways that decrease oxidative injury, inflammation, and apoptosis, while increasing vasculogenesis and neurogenesis. To determine the effects of rEpo on gene expression in 10-d-old BALB-c mice with unilateral brain injury, we compared microarrays from the hippocampi of brain-injured pups treated with saline or rEpo to similarly treated sham animals. Total RNA was extracted 24 h after brain injury and analyzed using Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. We identified sex-specific differences in hippocampal gene expression after brain injury and after high-dose rEpo treatment using single-gene and gene set analysis. Although high-dose rEpo had minimal effects on hippocampal gene expression in shams, at 24-h post brain injury, high-dose rEpo treatment significantly decreased the proinflammatory and antiapoptotic response noted in saline-treated brain-injured comparison animals.

Recombinant erythropoietin is neuroprotective in a novel mouse oxidative injury model.

To identify neuroprotective changes in gene expression, we developed a neonatal mouse model of moderate to severe oxidative brain injury and hypothesized that recombinant erythropoietin (rEpo) would decrease the expression of proapoptotic and proinflammatory genes 24 and 48 h, respectively, after injury and increase the expression of neurogenic and angiogenic genes 168 h after injury. Postnatal day 10 BALB-c mice underwent sham surgery or right common carotid artery occlusion followed by alternating hypoxia and hyperoxia and were then treated with rEpo (5,000 U/kg s.c.) or saline (vehicle) daily for up to three doses. At death, gross brain injury was assessed, then hippocampus, cortex, and thalamus were isolated for RNA or protein extraction. Microarray analysis, real-time polymerase chain reaction, and Bio-Plex suspension array system validation were performed. rEpo decreased both incidence and severity of brain injury (median injury score 3 vs. 0, p < 0.0001) and reduced the injury-induced increases in interleukin-1alpha and interleukin-6 gene expression (p < 0.001), with corresponding effects on protein translation. Similarly, the expression of caspase-1, caspase-4, and caspase-6 and of p53 was increased by brain injury at 24 h, but mitigated by rEpo (p < 0.01). The interleukin-10 expression was higher in the rEpo-treated animals. Apoptotic and proinflammatory gene expression persisted for 168 h. There was no increase in angiogenic gene expression at the time points studied.

Recent trends in erythropoietin-mediated neuroprotection.

Fifteen years of evidence have established that the cytokine erythropoietin offers promise as a treatment for brain injury. In particular, neonatal brain injury may be reduced or prevented by early treatment with recombinant erythropoietin. Extreme prematurity and perinatal asphyxia are common conditions associated with poor neurodevelopmental outcomes including cerebral palsy, mental retardation, hearing or visual impairment, and attention deficit hyperactivity disorder. When high doses of erythropoietin are administered systemically, a small proportion crosses the blood-brain barrier and can protect against hypoxic-ischemic brain injury. In addition to other protective effects, erythropoietin can specifically protect dopaminergic neurons. Since reduced dopamine neurotransmission contributes to attention deficit hyperactivity disorder, this condition may be amenable to erythropoietin treatment. This review focuses on the potential application of erythropoietin as a neuroprotectant with regard to neurologic complications of extreme prematurity, including attention deficit hyperactivity disorder. Recent concerns that early erythropoietin might exacerbate the pathologic neovascularization associated with retinopathy of prematurity are addressed.

Prenatal cord clamping in newborn Macaca nemestrina: a model of perinatal asphyxia.

Our objective was to establish a nonhuman primate model of perinatal asphyxia appropriate for preclinical evaluation of neuroprotective treatment strategies under conditions that closely resemble human neonatal emergencies, and to begin testing the safety and efficacy of erythropoietin neuroprotective treatment. Prior to delivery by hysterotomy, the umbilical cords of near term Macaca nemestrina (n = 8) were clamped for times ranging between 12 and 15 min. Animals received erythropoietin (5,000 U/kg/dose x 2 i.v., n = 3), or vehicle (n = 5) after resuscitation. We assessed physiologic parameters, continuous electroencephalogram, magnetic resonance imaging/spectroscopy, safety parameters and behavior. Animals were euthanized at 4 months of age. Mean birth weight was 507 +/- 62 g. Initial arterial pH ranged from 6.75 to 7.12, with base deficits of 17-25 mEq. Animals were flaccid at birth, with attenuated electroencephalograms, and seizures occurred in 3 of 8 animals. We demonstrated magnetic resonance imaging/spectroscopy changes consistent with hypoxia (elevated lactate levels were present in some animals), significant motor and behavioral abnormalities (particularly with 15 min of cord clamping), and evidence of gliosis at the time of death. We have established a reproducible model of moderate to severe perinatal hypoxic-ischemic injury in M. nemestrina newborns. This model, which combines structural, biochemical, and behavioral assessments over time can be used to assess the safety and efficacy of neuroprotective strategies.

High-dose erythropoietin inhibits apoptosis and stimulates proliferation in neonatal rat intestine.

BACKGROUND: Erythropoietin (Epo) receptors are widely expressed in the small bowel of neonatal rats and evidence suggests Epo has important trophic effects in developing bowel. OBJECTIVE: To compliment in vitro data, we directly examine in vivo the hypotheses that systemic Epo treatment can promote cell division and enterocyte migration, and arrest apoptosis in the ileum of neonatal rats. DESIGN: Epo (5000 U/kg s.c.) or vehicle treatments were given to one week old Sprague-Dawley rats (n = 86) along with timed injections of the thymidine analog 5-bromo-2-deoxyuridine (BrdU, 50mg/kg s.c.) to label DNA synthesis and track newly proliferating cells. To characterize the time course of effects, animals were killed at scheduled times from 30 min to 24 h after treatment. BrdU-containing cells were immunostained and counted in intestinal crypts, villi, and muscle wall of ileum. Effects of Epo on apoptosis were analyzed by TUNEL staining. Calibrated measurements were made to determine the density or relative proportion of BrdU- and TUNEL-positive cells. RESULTS: Systemic high-dose Epo promoted cell division in intestinal smooth muscle and enterocytes, stimulated migration of intestinal epithelial cells, and arrested apoptosis of enterocytes at the villous tips. CONCLUSION: These data provide in vivo evidence that Epo functions trophically in developing intestine tissues.

Patterns of thrombocytosis and thrombocytopenia in hospitalized neonates.

OBJECTIVE: We sought to identify changes in platelet (PLT) counts over time, and to evaluate the patterns of thrombocytopenia and thrombocytosis in hospitalized infants 23.8 weeks to term gestation. STUDY DESIGN: Neonates were divided into four gestational age groups and their PLT counts were retrospectively compared for prevalence of thrombocytopenia, thrombocytosis, and associated conditions. RESULTS: Postconceptional age, postnatal age, and sepsis (among other factors) affected PLT counts. When counts from noninfected appropriately grown infants were evaluated, the risk of thrombocytopenia and thrombocytosis were highest in the most preterm infants, and these risks changed with corrected gestational age. PLT counts increased weekly over the first 4 weeks of life for all but the most preterm infants. CONCLUSIONS: These data characterize the incidence of thrombocytopenia and thrombocytosis across a wide range of gestational ages and show that, even in noninfected neonates, these conditions are common, and risk decreases with increasing maturity. The age-related changes in PLT patterns may reflect maturation of platelet regulation.

Evaluation of eosinophilia in hospitalized preterm infants.

OBJECTIVE: To characterize changes in eosinophil counts over time, and to draw comparisons across four gestational age groups with respect to the incidence of, and clinical conditions associated with eosinophilia over the first months of life in a single neonatal intensive care unit (NICU). STUDY DESIGN: Weekly complete blood counts (CBCs) were collected from all NICU patients. Eosinophilia was classified as mild, moderate, or severe. Changes in eosinophil counts were examined over time, the incidence of eosinophilia was determined by gestational age group, and clinical correlations sought. RESULTS: Eosinophil data from 1652 CBCs were analyzed from 332 infants. Eosinophilia occurred in 148 infants (45%) during their hospitalization. The prevalence, severity, and timing of eosinophilia varied by gestational age, infection, and red blood cell transfusions. CONCLUSIONS: The incidence and severity of eosinophilia increased with immaturity, and was temporally associated with infection, necrotizing enterocolitis, and packed red blood cell transfusion.

Dose-dependent effects of morphine exposure on mRNA and microRNA (miR) expression in hippocampus of stressed neonatal mice.

Morphine is used to sedate critically ill infants to treat painful or stressful conditions associated with intensive care. Whether neonatal morphine exposure affects microRNA (miR) expression and thereby alters mRNA regulation is unknown. We tested the hypothesis that repeated morphine treatment in stress-exposed neonatal mice alters hippocampal mRNA and miR expression. C57BL/6 male mice were treated from postnatal day (P) 5 to P9 with morphine sulfate at 2 or 5 mg/kg ip twice daily and then exposed to stress consisting of hypoxia (100% N2 1 min and 100% O2 5 min) followed by 2h maternal separation. Control mice were untreated and dam-reared. mRNA and miR expression profiling was performed on hippocampal tissues at P9. Overall, 2 and 5 mg/kg morphine treatment altered expression of a total of 150 transcripts (>1.5 fold change, P<0.05) from which 100 unique mRNAs were recognized (21 genes were up- and 79 genes were down-regulated), and 5 mg/kg morphine affected 63 mRNAs exclusively. The most upregulated mRNAs were fidgetin, arginine vasopressin, and resistin-like alpha, and the most down-regulated were defensin beta 11, aquaporin 1, calmodulin-like 4, chloride intracellular channel 6, and claudin 2. Gene Set Enrichment Analysis revealed that morphine treatment affected pathways related to cell cycle, membrane function, signaling, metabolism, cell death, transcriptional regulation, and immune response. Morphine decreased expression of miR-204-5p, miR-455-3p, miR-448-5p, and miR-574-3p. Nine morphine-responsive mRNAs that are involved in neurodevelopment, neurotransmission, and inflammation are predicted targets of the aforementioned differentially expressed miRs. These data establish that morphine produces dose-dependent changes in both hippocampal mRNA and miR expression in stressed neonatal mice. If permanent, morphine-mediated neuroepigenetic effects may affect long-term hippocampal function, and this provides a mechanism for the neonatal morphine-related impairment of adult learning.

Evaluation of neutropenia and neutrophilia in hospitalized preterm infants.

OBJECTIVES: To characterize the time course of changes in neutrophil counts, and to draw comparisons across four gestational age groups with respect to incidence of neutropenia and neutrophilia over the first months of life in a single center. STUDY DESIGN: Weekly complete blood counts (CBC) were collected on all NICU patients. Changes in neutrophil counts over time were examined, the incidence of neutropenia (< or =1100/mm3) and neutrophilia (> or =15,000/mm3 before, and > or =6000/mm3 after 60 hours of life) were determined, and clinical correlations sought. RESULTS: A total of 2038 CBCs from 347 infants were evaluated. Changes in neonatal neutrophil counts were associated with gestational age, postnatal age, pregnancy-induced hypertension (PIH), and infection, either presumed or documented. Overall, neutropenia occurred 91 times in 50 infants (14% of infants), and the prevalence decreased with maturity and increased with exposure to PIH. In the first week of life, 69% of the neutropenia occurred in infants exposed to PIH. Neutrophilia occurred 579 times in 146 infants (42% of infants), and the prevalence decreased with maturity. Neutrophil deviations from the normal range were often asymptomatic and not associated with recognized clinical conditions. CONCLUSIONS: The incidence and prevalence of neutrophil abnormalities increased with immaturity, and were more common than previously appreciated in hospitalized preterm infants.

Adult responses to an ischemic stroke in a rat model of neonatal stress and morphine treatment.

Critically ill newborn infants experience stressors that may alter brain development. Using a rodent model, we previously showed that neonatal stress, morphine, and stress plus morphine treatments each influence early gene expression and may impair neurodevelopment and learning behavior. We hypothesized that the combination of neonatal stress with morphine may alter neonatal angiogenesis and/or adult cerebral blood vessel density and thus increase injury after cerebral ischemia in adulthood. To test this, neonatal Lewis rats underwent 8 h/d maternal separation, plus morning/afternoon hypoxia exposure and either saline or morphine treatment (2 mg/kg s.c.) from postnatal day 3-7. A subset received bromodeoxyuridine to track angiogenesis. Adult brains were stained with collagen IV to quantify cerebral blood vessel density. To examine vulnerability to brain injury, postnatal day 80 adult rats underwent right middle cerebral artery occlusion (MCAO) to produce unilateral ischemic lesions. Brains were removed and processed for histology 48 h after injury. Brain injury was assessed by histological evaluation of hematoxylin and eosin, and silver staining. In contrast to our hypothesis, neither neonatal morphine, stress, nor the combination affected cerebral vessel density or MCAO-induced brain injury. Neonatal angiogenesis was not detected in adult rats possibly due to turnover of endothelial cells. Although unrelated to angiogenesis, hippocampal granule cell neurogenesis was detected and there was a trend (P = 0.073) toward increased bromodeoxyuridine incorporation in rats that underwent neonatal stress. These findings are discussed in contrast to other data concerning the effects of morphine on cerebrovascular function, and acute effects of morphine on hippocampal neurogenesis.