Melatonin Alters Innate Immune Function in Infants with Neonatal Encephalopathy.

INTRODUCTION: Melatonin has been suggested an adjunctive therapy in neonatal encephalopathy (NE). Melatonin reduces oxidative stress and neutrophil activation; however, the immunological effects in NE have not been studied. METHODS: Infants with NE and neonatal controls were prospectively recruited. Whole blood was sampled in the first week of life. Following endotoxin and or melatonin treatment, diurnal variation was measured by RT PCR for circadian rhythm genes (brain and Muscle Arnt-Like protein [BMAL1], circadian locomotor output cycles kaput [CLOCK], Nuclear Receptor Subfamily 1 Group D Member 2 [REV Erß], and cryptochrome circadian clock [CRY]). Neutrophil and monocyte cell surface markers of activation CD11b, reactive oxygen intermediates (ROIs), and Toll-like receptor (TLR)-4 were also examined by flow cytometry in matching samples. RESULTS: Serum and RNA samples from forty infants were included (controls n = 20; NE n = 20) over the first week of life. Melatonin reduced neutrophil CD11b and TLR-4 expression in response to LPS in infants with NE compared to controls. There were no differences in ROIs. BMAL1 and CLOCK baseline gene expression levels were similar. BMAL1 was significantly decreased with LPS stimulation in NE. There was no significant diurnal variation in melatonin, neutrophil, and monocyte function or circadian genes. CONCLUSIONS: Melatonin alters immune function ex vivo in infants with NE. Infants with NE have altered immune circadian responses following LPS stimulation, which have potential for modulation.

Neonatal encephalopathy plasma metabolites are associated with neurodevelopmental outcomes.

BACKGROUND: To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. METHODS: Plasma samples from the NE (n = 45, day of life 0-1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). RESULTS: Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. CONCLUSIONS: Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors. IMPACT: Plasma metabolites may help to predict neurological outcomes in NE and supplement current clinical predictors. Current metabolomics research is limited in terms of clinical application and association with long-term outcomes. Our study presents novel associations of plasma metabolites from the first 24 h of life and 2-year neurodevelopmental outcomes for infants with NE. Our metabolomics discovery provides insight into possible disease mechanisms and methods to rescue and/or supplement metabolic pathways involved in NE. Our metabolomics discovery of metabolic pathway supplementations and/or rescue mechanisms may serve as adjunctive therapies for NE.

Multi-organ dysfunction scoring in neonatal encephalopathy (MODE Score) and neurodevelopmental outcomes.

AIM: Neonatal encephalopathy (NE) is associated with an increased risk of multi-organ injury. The lack of standardised definitions for multi-organ dysfunction in NE hinders accurate quantification of these complications. METHODS: A simple multi-organ dysfunction in neonatal encephalopathy scoring (MODE) system was created to include the cardiovascular, respiratory, gastrointestinal, haematological and neurological systems with a maximum score of 15. The MODE score was then compared with the grade of NE, Bayley Scales of Infant Development (Bayley-III) at 2 years of age and mortality. The Bayley score was used as it gave an objective score making it easier to compare the MODE score. Bayley score of <90 and/or abnormal MRI as an adverse outcome. RESULTS: Infants with perinatal asphyxia (PA:n = 85) were prospectively enrolled (PA only n = 9; NE I = 23; NE II = 42; NE III = 11). Infants with higher MODE scores were significantly more likely to have moderate/severe NE (NE II/III: median scores (IQR) 7(5-10) versus mild NE 2 (1-3); p-value < 0.001) The MODE score was highly predictive of mortality (AUC 0.96, p-value = 0.002). Infants who had an abnormal neurological examination at discharge or abnormal Bayley-III scores had significantly higher MODE scores (p-value = 0.001). CONCLUSION: Quantifying multi-organ injury is important to plan optimal early management and long-term follow-up. Additional use of clinical biomarkers may be useful as surrogate endpoints in future clinical trials and link to multi-organ longer-term developmental follow-up.

Plasma and Cerebrospinal Fluid Candidate Biomarkers of Neonatal Encephalopathy Severity and Neurodevelopmental Outcomes.

OBJECTIVES: To identify candidate biomarkers in both plasma and cerebrospinal fluid (CSF) that are associated with neonatal encephalopathy severity measured by encephalopathy grade, seizures, brain injury by magnetic resonance imaging (MRI), and neurodevelopmental outcomes at 15-30 months. STUDY DESIGN: A retrospective cohort study of plasma (N = 155, day of life 0-1) and CSF (n = 30, day of life 0-7) from neonates with neonatal encephalopathy and healthy neonates born at term (N = 30, ≥36 weeks of gestation) was conducted. We measured central nervous system necrosis (glial fibrillary acidic protein [GFAP], neurogranin [NRGN], tau), inflammatory (interleukin [IL]-6, IL-8, IL-10), and trophic (brain-derived neurotrophic factor [BDNF], vascular endothelial growth factor) proteins. Clinical outcomes were Sarnat scores of encephalopathy, seizures, MRI scores, and Bayley Scales of Infant and Toddler Development III at 15-30 months. RESULTS: Plasma NRGN, tau, IL-6, IL-8, and IL-10 were greater, whereas BDNF and vascular endothelial growth factor were lower in patients with neonatal encephalopathy vs controls. In plasma, tau, GFAP, and NRGN were directly and BDNF inversely associated with encephalopathy grade. IL-6 was inversely related to seizures. Tau was directly related to MRI abnormalities. Tau was inversely associated with Bayley Scales of Infant and Toddler Development III cognitive and motor outcomes. In CSF, NRGN was inversely associated with cognitive, motor, and language measures. GFAP, IL-6, and IL-10 were inversely related to cognitive and motor outcomes. IL-8 was inversely related to motor outcomes. CSF candidate biomarkers showed no significant relationships with encephalopathy grade, seizures, or MRI abnormalities. CONCLUSIONS: Plasma candidate biomarkers predicted encephalopathy severity, seizures, MRI abnormalities, and neurodevelopmental outcomes at 15-30 months.

Troponin T correlates with MRI results in neonatal encephalopathy.

AIM: Troponin is a sensitive marker of asphyxia in term infants mirroring the myocardial injury sustained in global hypoxia-ischaemia. In addition, troponin is a sensitive marker of severity of stroke in adults and neonatal encephalopathy (NE). We aimed to examine the relationship between troponin T in infants with perinatal asphyxia and brain injury on MRI and correlate with neurodevelopmental outcome. METHODS: Serum troponin was sampled in infants requiring resuscitation at birth and/or neonatal encephalopathy in a tertiary referral neonatal centre. Birth history, clinical parameters, neuroimaging and developmental outcome (Bayley Scores of Infant Development [BSID] III) were evaluated. RESULTS: Infants with perinatal asphyxia (n = 54) had serum troponin T measured and 27 required therapeutic hypothermia. Troponin T levels on days 1 and 2 were predictive of need for TH, development of seizures and grade II/III NE (AUC = 0.7; P-values < .001), troponin T levels on days 1, 2 and 3 were highly significant predictors of mortality (AUC = 0.99, P-values .005). The cut-off values of troponin T for best prediction of mortality were 0.84, 0.63 and 0.58 ng/mL on days 1, 2 and 3, respectively. Troponin T on day 3 of life was predictive of injury in the combined area of basal ganglia/watershed on MRI (AUC 0.70; P-value = .045). CONCLUSION: Infants with brain injury on neuroimaging following perinatal asphyxia had significantly elevated serum troponin, and troponin also correlated with developmental scores at 2 years. Further studies combining troponin and MRI may assist in the classification of neonatal brain injury to define aetiology, prognosis and response to treatment.

Neonatal Encephalopathy Is Associated With Altered IL-8 and GM-CSF Which Correlates With Outcomes.

Aim: To investigate the relationship between cytokines associated with innate immune cell activation and brain injury and outcome in infants with NE compared to neonatal controls. Methods: Serum and CSF biomarkers associated with activated neutrophils and monocytes [Interleukin-8 (IL-8) and Granulocyte-Macrophage-Colony-Stimulating-Factor (GM-CSF)] were serially measured using duplex immunoassays on days 1, 3 and 7 in term newborns with NE and controls. Results were compared to grade of encephalopathy, seizures, MRI brain imaging, mortality and Bayley Score of Infant and Toddler Development (Bayley-III) at 2 years of age. Results: Ninety-four infants had serum samples collected with 34 CSF samples. NE Grade II/III was significantly associated with elevated on day 2 serum IL-8. Mortality was best predicted by elevated day 1 IL-8. GM-CSF was initially elevated on day 1 and abnormal MRI imaging was associated with decreased day 2 GM-CSF. Elevated GM-CSF at day of life 6-7 correlated negatively with composite cognitive, language and motor Bayley-III scores at 2 years. Conclusion: Moderate or severe NE and mortality was associated with elevated IL-8. Day 2 GM-CSF could predict abnormal MRI results in NE and Bayley-III. Therefore, these cytokines are altered in NE and may predict early outcomes and further implicate inflammatory processes in NE.

Randomised trial of estimating oral endotracheal tube insertion depth in newborns using suprasternal palpation of the tip or weight.

BACKGROUND: Endotracheal tube (ETT) tip position is determined on chest X-ray (CXR) and should lie between the upper border of the first thoracic vertebra (T1) and the lower border of second thoracic vertebra (T2). Infant weight is commonly used to estimate how far the ETT should be inserted but frequently results in malpositioned ETT tips. Palpation of the ETT tip at the suprasternal notch has been recommended as an alternative. OBJECTIVE: To determine whether estimating ETT insertion depth using suprasternal palpation of the ETT tip rather than weight results in more correctly positioned ETT tips. DESIGN: Single-centre randomised controlled trial. SETTING: Level III neonatal intensive care unit (NICU) at a university maternity hospital. PATIENTS: Newborn infants without congenital anomalies intubated in the NICU. INTERVENTIONS: Participants were randomised to have ETT insertion depth estimated using palpation of the ETT tip at the suprasternal notch or weight [insertion depth (cm)=6 + wt (kg)]. MAIN OUTCOME MEASURE: Correct ETT position, that is, between the upper border of T1 and lower border of T2 on CXR, determined by one consultant paediatric radiologist masked to group assignment. RESULTS: There was no difference in the proportion of correctly placed ETT tips between the groups (suprasternal palpation 27/58 (47%) vs weight 23/60 (38%), p=0.456). Most incorrectly positioned ETTs were too low (56/68 (82%)). CONCLUSION: Estimating ETT insertion depth using suprasternal palpation did not result in more correctly positioned ETTs. TRIAL REGISTRATION NUMBER: ISRCTN13570106.

Coagulation Profiles Are Associated With Early Clinical Outcomes in Neonatal Encephalopathy.

Introduction: Neonatal encephalopathy (NE) is associated with coagulation abnormalities. We aimed to investigate the serial alterations in coagulation profiles in term infants with NE and correlate with their clinical outcomes. This was a prospective cohort study in a tertiary referral, university-affiliated maternity hospital. Neonates exposed to perinatal asphyxia were recruited (n = 82) and 39 received therapeutic hypothermia. Infants had serial coagulation tests including platelets, prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen in the first week of life. The main outcome measures included MRI brain and EEG seizures. Our results show that mortality was predicted on day 1 by decreased Fibrinogen (AUC = 0.95, p = 0.009) and by PT on day 2 with a cutoff of 22 s. An abnormal MRI was predicted by Fibrinogen on day 3 with a cut-off value of 2 g/L. For prediction of grade II/III NE, PT on day 2 of life was strongest with a cut-off value of 14 s. Only elevated APTT levels on day 1 of life were predictive of seizures (AUC = 0.65, p = 0.04). Conclusion: Coagulation parameters are strong predictors of outcomes such as abnormal NE grade, seizures, and mortality.

Serial cytokine alterations and abnormal neuroimaging in newborn infants with encephalopathy.

AIM: Inflammatory cytokines may play a role in the final common pathway in the pathogenesis of hypoxic-ischaemic injury in experimental models. We aimed to profile the systemic pro-and anti-inflammatory response over the first week of life in term infants at risk of neonatal encephalopathy. METHOD: In a tertiary referral university neonatal intensive care unit, serial blood samples were analysed from 41 term infants (requiring resuscitation at birth) in this prospective observational pilot study. Serum levels of 10 pro-and anti-inflammatory cytokines were evaluated including interleukin(IL)-1α, IL-1ß, IL-6, IL-8, IL-10, tumour necrosis factor(TNF)-α, interferon (IFN)-γ, vascular endothelial growth factor (VEGF), granulocyte/colony-stimulating factor (G-CSF) and granulocyte macrophage/colony-stimulating factor (GM-CSF). RESULTS: Infants with neonatal encephalopathy and abnormal neuroimaging (n = 15) had significantly elevated granulocyte macrophage/colony-stimulating factor at 0-24 h and interleukin-8, interleukin-6 and interleukin-10 at 24-48 hour. Tumour necrosis factor-α and vascular endothelial growth factor levels were lower at 72-96 hour (p < 0.05). Significantly elevated levels of interleukin-10 were associated with mortality. CONCLUSION: Serum cytokine changes and innate immune dysregulation in the first week of life may be indicators of outcome in neonatal encephalopathy but require validation in larger studies.

Persistent systemic monocyte and neutrophil activation in neonatal encephalopathy.

AIM: Circulating immune cell activation is associated with worse outcome in adult and animal models of brain injury. Our aim was to profile the systemic inflammatory response over the first week of life in infants at risk of neonatal encephalopathy (NE) and correlate early neutrophil and monocyte endotoxin and activation responses with outcome. METHODS: Prospective observational study in a tertiary referral university hospital including 22 infants requiring resuscitation at birth who had serial (five time points) neutrophil and monocyte CD11b (marker of cell adhesion), intracellular reactive oxygen intermediates (ROI; cell activation) and Toll-like receptor (TLR; endotoxin recognition) before and after endotoxin stimulation ex vivo compared to neonatal controls. RESULTS: All neonates requiring resuscitation at delivery (n = 122 samples) had higher neutrophil and monocyte CD11b and TLR-4 expression compared with adults and neonatal controls. Neonates with abnormal neuroimaging and/or severe NE had increased CD11b, ROI and TLR-4. Increased polymorphonuclear leukocytes TLR-4 expression was associated with increased mortality in infants with NE. CONCLUSION: Innate immune dysregulation in the first week of life is associated with severity of outcome in neonatal brain injury in this cohort and may be amenable to immunomodulation.

Neutrophil and monocyte toll-like receptor 4, CD11b and reactive oxygen intermediates, and neuroimaging outcomes in preterm infants.

BACKGROUND: Activated leukocytes and infection are implicated in neonatal brain injury. Leukocyte surface receptors are increased in stroke models and may be targets for future adjunctive therapies. METHODS: Serial blood samples were analyzed from preterm infants (n = 51; <32 wk gestation) on days 0, 1, 2, and 7 of life. Monocyte and neutrophil activation were evaluated via flow cytometry at baseline and following endotoxin stimulation ex vivo by measuring CD11b (activation), toll-like receptor 4 (TLR-4; endotoxin recognition) expression, and intracellular reactive oxygen intermediate (ROI) production (function). RESULTS: Control preterm infants with normal neuroimaging had elevated baseline CD11b and TLR-4 expression and ROI production compared with adults as well as a robust immune response following endotoxin stimulation. Preterm infants with abnormal neuroimaging had increased neutrophil TLR-4 and ROI compared with all controls. CONCLUSION: Preterm infants have a robust immune response compared with adults. Increased TLR-4 expression in preterm infants with abnormal neuroimaging is similar to findings in adult stroke. In addition, ROI production may cause tissue injury. The modulation of these responses may be beneficial in preterm inflammatory disorders.

Easily missed, potentially fatal complication in an extremely preterm infant.

A baby girl was delivered by emergency caesarean section at 23+6 weeks gestation weighing 440 g. Apgar scores were 1, 3 and 4 at 1, 5 and 10 min, respectively. She was intubated and transferred to the neonatal intensive care unit. Umbilical arterial and venous lines and an orogastric tube (OGT) were inserted. On day 4 of life the OGT appeared to be outside of the gastrointestinal tract on X-ray. Feeds were held and contrast oesophagography confirmed suspicion of an oesophageal perforation. She was treated with intravenous metronidazole, gentamycin and amoxicillin and placed nil by mouth for 10 days. Resolution of the perforation was confirmed on repeat contrast study (day 10) and feeds were restarted with no further complications.

Isolated acute non-cystic white matter injury in term infants presenting with neonatal encephalopathy.

We discuss possible aetiological factors, MRI evolution of injury and neuro-developmental outcomes of neonatal encephalopathy (NE). Thirty-six consecutive infants diagnosed with NE were included. In this cohort, four infants (11%) were identified with injury predominantly in the deep white matter on MRI who were significantly of younger gestation, lower birthweight with higher Apgars at one and five minutes compared to controls. Placental high grade villitis of unknown aetiology (VUA) was identified in all four of these infants. Our hypothesis states VUA may induce white matter injury by causing a local inflammatory response and/or oxidative stress during the perinatal period. We underline the importance of continued close and systematic evaluation of all cases of NE, including examination of the placenta, in order to come to a better understanding of the clinical presentation, the patterns of brain injury and the underlying pathophysiological processes.

Radiological versus clinical evidence of malrotation, a tortuous tale--10-year review.

INTRODUCTION: Malrotation is a common abnormality, often diagnosed in the neonatal period. Symptoms may be nonspecific and clinical signs of volvulus are often only seen in the late stages when there has been significant ischemic insult to the bowel. The gold standard diagnostic investigation is the upper gastrointestinal (UGI) contrast study. This study was designed to assess the incidence of negative laparotomy in patients with malrotation diagnosed on UGI contrast study and to identify the clinical signs and symptoms at presentation. METHODS: A retrospective review of patients who underwent laparotomy for malrotation, over a 10-year period (2001 to 2010) was performed. Inclusion criteria were patients<16 years of age with a diagnosis of malrotation on preoperative UGI contrast study. RESULTS: A total of 72 patients were reviewed. UGI contrast study diagnosed 43(60%) cases of malrotation without volvulus, and 29(40%) cases of malrotation with volvulus. Laparotomy revealed that 39(54.2%) patients had malrotation without volvulus, 27(37.5%) had malrotation with volvulus, and 6(8.3%) had no evidence of malrotation. A total of 13(18%) patients had an incorrect diagnosis on UGI contrast study; 6(8.3%) had normal anatomy, 3(4.2%) diagnosed with malrotation without volvulus, had volvulus at laparotomy, and 4(5.5%) diagnosed with malrotation and volvulus had no volvulus at laparotomy. CONCLUSION: UGI contrast study can occasionally be misleading. In the above study, we have shown that there is a significant rate of negative laparotomy following diagnosis of malrotation on UGI contrast study. Therefore, we would advocate that all parents of patients undergoing laparotomy for malrotation should be informed of the risk of negative laparotomy as part of the consent process.