Paediatrics & Child Health - Doctoral Theses

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Ability of early neurological assessment and continuous EEG to predict long term neurodevelopmental outcome at 5 years in infants following hypoxic-ischaemic encephalopathy
(University College Cork, 2018) O'Connor, Catherine M.; Murray, Deirdre M.; Boylan, Geraldine B.; Health Research Board
Hypoxic-ischaemic encephalopathy (HIE) symptoms evolve during the first days of life and their monitoring is critical for treatment decisions and long-term outcome predictions. This thesis aims to report the five-year outcome of a HIE cohort born in the pre-therapeutic hypothermia era and to evaluate the predictive value of (a) neonatal neurological and EEG markers and (b) development in the first 24 months, for outcome. Methods: Participants were recruited at age five from two birth cohorts; HIE and Comparison. Repeated neonatal neurological assessments using the Amiel-TisonNeurological-Assessment-at-Term, continuous video EEG monitoring in the first 72 hours, and Sarnat grading at 24 hours were recorded. EEG severity grades were assigned at 6, 12 and 24 hours. Development was assessed in the HIE cohort at 6, 12 and 24 months using the Griffiths Mental Development (0-2) Revised Scales. At age five, intellectual (WPPSI-IIIUK scale), neuropsychological (NEPSY-II scales), neurological and ophthalmic testing was completed. Results: 5-year outcomes were available for 81.5% (n=53) of HIE and 71.4% (n=30) of Comparison cohorts. In HIE, 47.2% (27% mild, 47% moderate, 83% severe Sarnat), had non-intact outcome vs. 3.3% of the Comparison cohort. Non-intact outcome rates by 6-hour EEG-grade were: grade0=3%, grade1=25%, grade2=54%, grade3/4=79%. In HIE, processing speed (p=0.01) and verbal short-term memory (p=0.005) were below test norms. No significant differences were found in IQ, NEPSY-II or ocular biometry scores between children following mild and moderate HIE. Median IQ scores for mild (99(94-112),p=grade 2) at 24hours had superior positive predictive value (74%; AUROC(95%CI)=0.70(0.55-0.85) for non-intact 5-year outcome than abnormal EEG at 6 hours (68%; AUROC(95%CI)=0.71(0.56-0.87). Within-child development scores were inconsistent across the first 24 months. Although all children with intact 24-month Griffiths quotient (n=30) had intact 5-year IQ, 8/30 had non-intact overall outcome. Conclusion: Predictive value of neonatal neurological assessments and an EEG grading system for outcome was confirmed. Intact early childhood outcomes post-HIE may mask subtle adverse neuropsychological sequelae into the school years. This thesis supports emerging evidence that mild-grade HIE is not a benign condition and its inclusion in studies of neuroprotective treatments for HIE is warranted.
Altered cord blood biomarkers in neonatal brain injury
(University College Cork, 2019) O'Sullivan, Marc Paul; Murray, Deirdre M.; Boylan, Geraldine B.; Clarke, Gerard; National Children's Research Centre; Health Research Board
Introduction: Alterations in RNA and proteins observed at birth may have long-term effects on early life. Perinatal asphyxia (PA) accounts for a quarter of all global neonatal deaths. Early intervention is critical for developing strategies to improve long-term outcome. Early identifiers for infants at risk of HIE is prone to subjective-bias or poorly objective biochemical measurements. The umbilical cord blood (UCB) can give a snapshot of both the newborn and the in-utero environment. It is non-invasive to both mother and infant and contains microRNA (miRNA), messenger RNA (mRNA) and circulating proteins that can exist stably in the circulation. This thesis aims to progress further the knowledge of UCB biomarkers using low-throughput techniques in both RNA and protein analyses, and to explore and validate proposed and potential biomarkers in HIE. Methods: This thesis utilised three distinct, well-defined cohorts throughout, the BiHIVE1 cohort (2009- 2011), the BiHiVE2 cohort (2012-1015), and the BASELINE longitudinal birth cohort (2008- 2016). The BiHIVE1 cohort was recruited in Cork, Ireland (7500 live births per annum), this study included full-term infants with PA enrolled at birth. This cohort recruited 112 cases (40 developed HIE - 24 mild, 6 moderate and 10 severe), of these 52 had neurodevelopmental follow-up, and 7 died in infancy. For this cohort, healthy controls were recruited from the BASELINE cohort. The BiHiVE2 validation cohort was recruited in Cork, Ireland and Karolinska Huddinge, Sweden (4400 live births per annum), this study included infants with PA along with healthy control infants. This cohort recruited 353 cases (48 developed HIE - 32 mild, 14 moderate and 2 severe) and 289 controls, 449 had neurodevelopmental follow-up, and no deaths occurred in infancy. Infants with perinatal asphyxia had matched inclusion criteria across both BiHiVE1 and BiHiVE2. Infants were assigned a modified Sarnat score at 24 hours and were followed-up with neurological assessment and neurodevelopmental outcome at 18-36 months of age. Umbilical cord serum, plasma and whole blood were processed and biobanked across all cohorts at delivery and stored at -80°C. Techniques employed throughout the thesis include blood processing, RNA isolations of whole blood miRNA and mRNA, and subsequent cDNA synthesis and quantitative real-time polymerase chain reaction (qPCR). Protein expression measurements were conducted with sandwich enzyme-linked immunosorbent assays (ELISA) on cord serum samples. All statistical analyses were conducted using IBM SPSS Statistics 24, graphical representation of data was generated using GraphPad Prism 8. Results: miRNA Panel: One-hundred and sixty infants had miRNA qPCR analyses across both BiHiVE1 and BiHiVE2. In BiHIVE1, 12 candidate miRNAs were identified, and 7 of these miRNAs were chosen for validation in BiHIVE2. The BiHIVE2 cohort showed consistent alteration of 3 miRNAs; miR-374a-5p was decreased in infants diagnosed as having HIE compared with healthy control infants (P = 0.009), miR-376c-3p was decreased in infants with PA compared with healthy control infants (P = 0.004), and miR-181b-5p was decreased in infants eligible for therapeutic hypothermia (TH) (P = 0.02). Predicted mRNA Targets: One-hundred and twenty-six infants had mRNA analyses. In the grade of HIE severity, the level of mFZD4 was increased in severe HIE vs mild HIE (P = 0.004), and severe HIE vs moderate HIE (P = 0.003). Fifty-six infants were included in the neurodevelopmental outcome analysis; Levels of mNFAT5 were increased in severely abnormal vs normal outcome (P = 0.036), and in severely abnormal vs mildly abnormal outcome (P = 0.013). Levels of mFZD4 were increased in severely abnormal vs normal outcome (P = 0.004), and in severely abnormal vs mildly abnormal outcome (P = 0.026). Interleukin-16: One-hundred and thirteen infants were included in the final analyses. Cord blood-based IL-16 was increased in infants with perinatal asphyxia and HIE relative to controls (P = 0.025). IL-16 was also increased in the HIE group relative to controls (P = 0.042). There was no significant difference in IL-16 across grades of HIE or in those with abnormal outcomes at 2-years of age. Activin A and mACVR2B: Serum activin A analyses included 101 infants. No differences were observed across the groups (P = 0.693). The HIE group included 23 infants; a combination of mild and moderate HIE, without infants with a severe grade. No differences were observed across the grades of HIE (P = 0.115). Whole blood mACVR2B analyses included 68 infants, and no differences were observed across the groups (P = 0.746). The HIE group included 22 infants, and no differences were observed across the grades of HIE (mild and moderate only) (P = 0.468). No differences were observed in infants followed up to 18-36 months in serum activin A or in whole blood mACVR2B, (P = 0.550) and (P = 0.881) respectively. Conclusions: This thesis has identified and validated the decreased expression of UCB miRNA (miR-181b, 374a and miR-376c), increased expression of UCB mRNA (mNFAT5 and mFZD4) and increased UCB cytokine expression (IL-16) across two cohorts in HIE. It validates previous miRNA whole blood biomarkers of PA (miR-376c) and HIE (miR-374a) and proposes novel whole blood mRNA biomarkers as assessors for both HIE severity (mFZD4) and long-term neurodevelopmental outcome (mNFAT5 and mFZD4), which can outperform current measurements. These miRNA and mRNA could aid current measures as objective diagnostic and prognostic markers of HIE. It has further explored the strengths of cytokines and proteins and confirmed their inability as biomarkers across HIE groups and grades. This work has an important role in further developing UCB-based biomarkers in early life.
Early biomarkers to predict grade of encephalopathy following hypoxic ischaemic injury
(University College Cork, 2014) Walsh, Brian; Boylan, Geraldine B.; Kenny, Louise C.; Murray, Deirdre M.; Molecular Medicine Ireland; Higher Education Authority
The standard early markers for identifying and grading HIE severity, are not sufficient to ensure all children who would benefit from treatment are identified in a timely fashion. The aim of this thesis was to explore potential early biomarkers of HIE. Methods: To achieve this a cohort of infants with perinatal depression was prospectively recruited. All infants had cord blood samples drawn and biobanked, and were assessed with standardised neurological examination, and early continuous multi-channel EEG. Cord samples from a control cohort of healthy infants were used for comparison. Biomarkers studied included; multiple inflammatory proteins using multiplex assay; the metabolomics profile using LC/MS; and the miRNA profile using microarray. Results: Eighty five infants with perinatal depression were recruited. Analysis of inflammatory proteins consisted of exploratory analysis of 37 analytes conducted in a sub-population, followed by validation of all significantly altered analytes in the remaining population. IL-6 and IL-6 differed significantly in infants with a moderate/severely abnormal vs. a normal-mildly abnormal EEG in both cohorts (Exploratory: p=0.016, p=0.005: Validation: p=0.024, p=0.039; respectively). Metabolomic analysis demonstrated a perturbation in 29 metabolites. A Cross- validated Partial Least Square Discriminant Analysis model was developed, which accurately predicted HIE with an AUC of 0.92 (95% CI: 0.84-0.97). Analysis of the miRNA profile found 70 miRNA significantly altered between moderate/severely encephalopathic infants and controls. miRNA target prediction databases identified potential targets for the altered miRNA in pathways involved in cellular metabolism, cell cycle and apoptosis, cell signaling, and the inflammatory cascade. Conclusion: This thesis has demonstrated that the recruitment of a large cohortof asphyxiated infants, with cord blood carefully biobanked, and detailed early neurophysiological and clinical assessment recorded, is feasible. Additionally the results described, provide potential alternate and novel blood based biomarkers for the identification and assessment of HIE.
Early milk diet of infants and the effect on their body composition and growth and development in the first two years of life
(University College Cork, 2018) Smith, Hazel Ann; Murray, Deirdre M.; Leahy-Warren, Patricia; National Children's Research Centre (NCRC)
Background: Nutrition in the first few months of life has important effects on long-term growth. The aim of this PhD was to investigate the effect of an infant’s milk diet (both formula and breastmilk intake) in the first two months of life on body composition at two months of age, growth in the first two years of life and neurodevelopment at two years; and to examine whether breast- and formula-fed infants differ at birth, confounding the true effect of breastfeeding. Methods: Secondary data analysis of the feeding patterns, growth and development of children in the Cork BASELINE Birth Cohort Study. Descriptive and multivariate (multi-linear and logistic regression) analysis was employed. Results: Admission to the neonatal intensive care unit had the greatest negative impact on exclusively breastfeeding at two months (adjusted odds ratio = 0.20 (95% CI 0.05, 0.83)). Nearly twice as many exclusively formula-fed infants experienced early rapid growth (ERG) at two months compared to exclusively breastfed infants, n=87 (30%) vs n=56 (16.9%), respectively. Infants that experienced ERG saw an increase in their weight-for-height (wfh) z-score at 24 months compared to infants that did not experience ERG, β=0.39 (95% CI 0.19, 0.54). Breastfed infants had a higher mean(SD) birthweight to formula-fed infants, 3.56(0.42)kg versus 3.46(0.44)kg, respectively. However, breastfed infants had a lower mean(SD) percentage fat mass at birth compared to formula-fed infants, 10.01(3.71)% versus 12.05(4.06)%. Conclusion statement: By two months of age few Irish infants are exclusively breastfed. Formula supplementation and admission to the neonatal intensive care unit in the maternity hospital shortened breastfeeding duration. Formula feeding increased the odds of ERG and experiencing ERG at two months increased a child’s wfh z-score at 24 months. Breastfed infants were different in growth and body composition at birth in our cohort.
An exploration of the positive and negative relationships associated with the development of asthma and atopic disorders in primary school children in Cork
(University College Cork, 2013) Duggan, Eileen Mary; Hourihane, Jonathan O'B.; Fitzgerald, Tony; Irish Lung Foundation
Childhood asthma, allergic rhinitis and eczema are complex heterogenic chronic inflammatory allergic disorders which constitute a major burden to children, their families. The prevalence of childhood allergic disorders is increasing worldwide and merely rudimentary understanding exists regarding causality, or the influence of the environment on disease expression. Phase Three of the International Study of Asthma and Allergy in Childhood (ISAAC) reported that Irish adolescents had the 4th highest eczema and rhinoconjunctivitis prevalence and 3rd highest asthma prevalence in the world. There are no ISAAC data pertaining to young Irish children. In 2002, Sturley reported a high prevalence of current asthma in Cork primary school children aged 6-9 years. This thesis comprises of three cross-sectional studies which examined the prevalence of and associations with childhood allergy and a quasi-retrospective cohort study which observed the natural history of allergy from 6-9 until 11-13 years. Although not part of ISAAC, data was attained by parentally completed ISAAC-based questionnaires, using the ISAAC protocol. The prevalence, natural history and risk factors of childhood allergy in Ireland, as described in this thesis, echo those in worldwide allergy research. The variations of prevalence in different populations worldwide and the recurring themes of associations between childhood allergy and microbial exposures, from farming environments and/or gastrointestinal infections, as shown in this thesis, strengthen the mounting evidence that microbial exposure on GALT may hold the key to the mechanisms of allergy development. In this regard, probiotics may be an area of particular interest in allergy modification. Although their effects in relation to allergy, have been investigated now for several years, our knowledge of their diversity, complex functions and interactions with gut microflora, remain rudimentary. Birth cohort studies which include genomic and microbiomic research are recommended in order to examine the underlying mechanisms and the natural course of allergic diseases.
Heart rate variability and electroencephalography in Infants with hypoxic ischaemic encephalopathy
(University College Cork, 2017) Goulding, Robert Michael David; Boylan, Geraldine B.; Stevenson, Nathan J.; Filan, Peter M.; Science Foundation Ireland
Hypoxic-ischaemic encephalopathy (HIE) remains a significant cause of neurological injury in the newborn. HIE is associated with altered autonomic function. Heart rate variability (HRV) is a direct measure of autonomic control and this thesis aims to investigate the use of HRV as a reliable, routinely recorded physiological marker to identify HIE severity, seizure activity, and predict long-term outcome. Measures of HRV were calculated on a cohort of full-term infants with HIE, recruited from 2003-2012, prior to and following the introduction of therapeutic hypothermia (TH) in the neonatal intensive care unit. Simultaneous, 1h periods of EEG and ECG activity were recorded from 6h-90h after birth. EEG activity was used to grade epochs as mild, moderate, or severe. Normalised-RR intervals were used to calculate seven HRV features from the ECG signal: Mean (mean NN), standard deviation (SDNN), triangular interpolation (TINN), power in high frequency (HF), low frequency (LF), very low frequency bands (VLF), and LF/HF ratio. There were significant correlations between HRV and EEG grade in normothermic (r = -0.45 to -0.33; n=44) and hypothermic groups (r = -0.39 to -0.22 ; n=74). In infants with moderate HIE, there were significant differences between normothermic and hypothermic groups (HF: p=0.016, LF/HF ratio: p=0.006, mean NN: p<0.001). HRV was significantly associated with outcome in both normothermic and hypothermic groups (normothermic at 24h, 48h and hypothermic 12h, 24h, and 48h after birth). Infants with neonatal seizures and severe HIE had an increase in HRV during electrographic seizures (TINN: p= 0.034, VLF: p=0.028, LF: p=0.006, HF: p=0.015). The measurement of autonomic function with HRV has potential as a diagnostic and prognostic tool in infants with HIE.
Improving the care of preterm infants: before, during, and after, stabilisation in the delivery room
(University College Cork, 2015) Hawkes, Gavin; Dempsey, Eugene M.; Ryan, C. Anthony
Introduction Up to 10% of infants require stabilisation during transition to extrauterine life. Enhanced monitoring of cardiorespiratory parameters during this time may improve stabilisation outcomes. In addition, technology may facilitate improved preparation for delivery room stabilisation as well as NICU procedures, through educational techniques. Aim To improve infant care 1) before birth via improved training, 2) during stabilisation via enhanced physiological monitoring and improved practice, and 3) after delivery, in the neonatal intensive care unit (NICU), via improved procedural care. Methods A multifaceted approach was utilised including; a combination of questionnaire based surveys, mannequin-based investigations, prospective observational investigations, and a randomised controlled trial involving preterm infants less than 32 weeks in the delivery room. Forms of technology utilised included; different types of mannequins including a CO2 producing mannequin, qualitative end tidal CO2 (EtCO2) detectors, a bespoke quantitative EtCO2 detector, and annotated videos of infant stabilisation as well as NICU procedures Results Manual ventilation improved with the use of EtCO2 detection, and was positively assessed by trainees. Quantitative EtCO2 detection in the delivery room is feasible, EtCO2 increased over the first 4 minutes of life in preterm infants, and EtCO2 was higher in preterm infants who were intubated. Current methods of heart rate assessment were found to be unreliable. Electrocardiography (ECG) application warrants further evaluation. Perfusion index (PI) monitoring utilised in the delivery room was feasible. Video recording technology was utilised in several ways. This technology has many potential benefits, including debriefing and coaching in procedural healthcare, and warrants further evaluation. Parents would welcome the introduction of webcams in the NICU. Conclusions I have evaluated new methods of improving infant care before, during, and after stabilisation in the DR. Specifically, I have developed novel educational tools to facilitate training, and evaluated EtCO2, PI, and ECG during infant stabilisation. I have identified barriers in using webcams in the NICU, to now be addressed prior to webcam implementation.
An in-depth characterisation of neonatal seizures by early continuous video-EEG analysis
(University College Cork, 2016) Low, Evonne; Boylan, Geraldine B.; Ryan, C. Anthony; Wellcome Trust
Introduction Seizures are harmful to the neonatal brain; this compels many clinicians and researchers to persevere further in optimizing every aspects of managing neonatal seizures. Aims To delineate the seizure profile between non-cooled versus cooled neonates with hypoxic-ischaemic encephalopathy (HIE), in neonates with stroke, the response of seizure burden to phenobarbitone and to quantify the degree of electroclinical dissociation (ECD) of seizures. Methods The multichannel video-EEG was used in this research study as the gold standard to detect seizures, allowing accurate quantification of seizure burden to be ascertained in term neonates. The entire EEG recording for each neonate was independently reviewed by at least 1 experienced neurophysiologist. Data were expressed in medians and interquartile ranges. Linear mixed models results were presented as mean (95% confidence interval); p values <0.05 were deemed as significant. Results Seizure burden in cooled neonates was lower than in non-cooled neonates [60(39-224) vs 203(141-406) minutes; p=0.027]. Seizure burden was reduced in cooled neonates with moderate HIE [49(26-89) vs 162(97-262) minutes; p=0.020] when compared with severe HIE. In neonates with stroke, the background pattern showed suppression over the infarcted side and seizures demonstrated a characteristic pattern. Compared with 10 mg/kg, phenobarbitone doses at 20 mg/kg reduced seizure burden (p=0.004). Seizure burden was reduced within 1 hour of phenobarbitone administration [mean (95% confidence interval): -14(-20 to -8) minutes/hour; p<0.001], but seizures returned to pre-treatment levels within 4 hours (p=0.064). The ECD index in cooled, non-cooled neonates with HIE, stroke and in neonates with other diagnoses were 88%, 94%, 64% and 75% respectively. Conclusions Further research exploring the treatment effects on seizure burden in the neonatal brain is required. A change to our current treatment strategy is warranted as we continue to strive for more effective seizure control, anchored with use of the multichannel EEG as the surveillance tool.
Inflammation driven molecular alterations in disorders of the perinatal brain
(University College Cork, 2021-03) Casey, Sophie; Murray, Deirdre M.; O'Keeffe, Gerard W.; Boylan, Geraldine B.; Irish Research Council
Introduction: Inflammatory insults during the perinatal period are known to disrupt normal neurodevelopmental processes. Neonatal hypoxic ischemic encephalopathy (HIE) may occur with or without infection and is characterised by a significant immunoinflammatory response. Infection sensitises the neonatal brain to further hypoxic-ischemic injury. HIE with and without infection affects approximately 3 per thousand of all live births in the developed world. Despite the advent of therapeutic hypothermia (TH), almost half of affected neonates die or are left with lifelong disabilities or disorders. Autism Spectrum Disorder (ASD) is one such disorder and affects approximately 1.5% of the population in the developed world. Epidemiological and animal studies suggest that maternal immune dysregulation may contribute to the development of ASD. Rapid identification of neonates and infants at risk of HIE and ASD is vital, but many miss the critical therapeutic windows due to subjective and suboptimal diagnostic techniques. Circulating inflammation-associated markers such as microRNA (miRNA) and cytokines may hold the key to rapid diagnosis, providing an insight into maternal, foetal and neonatal injury cascades. Blood represents a non-invasive, and rapidly accessible medium in which to examine aberrant expression of these inflammation-driven molecular markers. In addition, miRNAs and cytokines are stably expressed in the blood, so may represent robust biomarker candidates. Furthermore, while many studies of miRNA expression have been performed in clinical cases of HIE, their functional role is still largely unknown. The primary hypothesis of this thesis is that examination of temporal inflammatory profiles may improve our understanding of the pathophysiology of early-life neuronal injury and subsequent development of long-term disabilities, and may provide novel biomarkers, therapeutic targets, or candidates capable of improving long-term outcomes. The current thesis aims to identify early molecular biomarkers of HIE and ASD in clinical and preclinical models and investigate the functional role of altered miRNAs in vitro. To investigate this, we aimed to examine temporal profiles of miRNA and cytokine alterations in a large animal model of HIE, with and without inflammatory sensitisation. Following this, we aimed to identify rapidly detectable miRNA and cytokine biomarkers of HIE, with and without inflammatory sensitisation. Lastly, we aimed to investigate the role of altered miRNA biomarkers through identification and examination of their functional targets, and to examine the mid-gestational cytokine profiles of mothers who give birth to offspring with ASD in a large clinical cohort. Methods: The current thesis utilised a large cohort of HIE neonates recruited to the Validation of Biomarkers in Hypoxic Ischemic Encephalopathy (BiHIVE) 1 & 2 studies (NCT02019147) and a clinically relevant large animal model of HIE with and without inflammation-sensitisation – the neonatal porcine model. To achieve a moderate-severe level of hypoxia-ischemia (HI) in piglets, inspired oxygen was reduced to 4% for approximately 30 minutes and titrated to achieve the desired level of neuronal injury. To achieve inflammation-sensitisation, piglets received an infusion of lipopolysaccharide (LPS) prior to HI. Blood was drawn from piglets at regular intervals for 48-72h following injury. Whole blood was used for analysis of miRNA content, while serum was preferable for analysis of cytokines. The multi-national Screening for Pregnancy Endpoints (SCOPE) cohort was utilised for the examination of circulating midgestational cytokine markers of ASD following diagnosis at neurodevelopmental follow-up. Cohorts from Cork, Ireland and Auckland, New Zealand were examined. Techniques employed throughout the current thesis are state-of-the-art, and were performed by both the PhD candidate and collaborating co-authors. Neurodevelopmental outcomes for neonatal study subjects were measured using Apgar/Sarnat staging, clinical signs (Chapter 3), early intervention services teams and child psychiatrists (Chapter 6). Outcomes for animal subjects were measured using clinical signs and Haemotoxylin & Eosin (H&E)/Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining of porcine brain tissues (Chapters 3 and 4). Further techniques featured in Chapter 3 include ribonucleic acid (RNA) Sequencing (RNA-Seq), Haemotoxylin & Eosin (H&E) staining of porcine brain tissues, magnetic resonance spectroscopy (MRS), RNA isolation from whole blood, complementary deoxyribonucleic acid (cDNA) synthesis, quantitative real-time polymerase chain reaction (qRT-PCR), in silico analysis, cell culture of SH-SY5Y cells and primary cultures of embryonic day (E) 14 rat midbrain, in vitro transfection of miRNA inhibitors, immunocytochemistry and fluorescent microscopy. Techniques used throughout the remainder of the thesis were performed solely by the PhD candidate. Techniques employed in Chapters 4 and 6 include electrochemiluminescent Mesoscale Discovery sandwich enzyme-linked immunosorbent (ELISA) immunoassays for analysis of serum cytokines. Finally, Chapter 5 used cell cultures of SH-SY5Y cells, in vitro transfection of miRNA inhibitors, green fluorescent protein (GFP) based reporter assays, immunocytochemistry, and fluorescent microscopy. All statistical analyses were conducted using IBM SPSS Statistics 24/26 and GraphPad Prism 7/8. Graphical representations of data were generated using GraphPad Prism 8. Results: miRNA Dysfunction in the Porcine Model of HIE Eleven candidate miRNAs were determined from a combination of results generated from microarray and RNA-Seq from a clinical cohort of hypoxic-ischemic neonates. These were then examined in the porcine model over 72h. Six miRNAs - miR-128, miR-148a, miR-151a, miR-181a, miR-181b and miR-374a, were upregulated in whole blood 1h after HI. Three - miR-374a, miR-181a and miR-181b, were specifically upregulated in moderate-severely injured piglets. miRNA levels at 1h correlated with histopathological and MRS-measured Lactate/Creatine (Lac/Cr) neuropathological outcomes. Inhibition of miR-181a in vitro resulted in increased neurite growth and increased expression of one member of the target bone morphogenetic protein (BMP) signalling pathway - BMPR2. Cytokine Dysfunction in the Porcine Model of Inflammation-Sensitised HIE Eight candidate cytokines were examined in serum samples from the inflammation-sensitised porcine model over 48h. Interleukin (IL) 6, tumor necrosis factor (TNF) α and Tau displayed a sustained inflammatory response following LPS exposure with and without hypoxia. Neuron-specific enolase (NSE) increased slowly following HI. LPS + Hypoxia-ischemia (LPS-HI) piglets displayed late increases in both NSE and C-reactive protein (CRP). TNFα and IL-6 allowed discrimination between animals who were exposed to inflammation-sensitised HI and those who were exposed to HI alone 6h following a moderate-severe HIE-like insult. miRNA Function In Vitro We previously identified the BMP signalling pathway as a predicted downstream target of miR-374a and miR-181a. The effect of miR-374a and miR-181a manipulation on the BMP-small mothers against decapentaplegic (SMAD) signalling pathway was investigated in vitro. miR-374a inhibition increased expression of BMP2 and BMPR2 but did not alter transcription of SMAD. Likewise, miR-181a inhibition was previously found to increase BMPR2 expression and did not alter SMAD transcription in vitro. Midgestational Cytokine Dysfunction in ASD Eight candidate cytokines were examined in serum samples retrieved at 15 and 20 weeks’ gestation in mothers of children affected by ASD. IL-17A concentrations were downregulated at 20 weeks’ gestation in mothers of children who progressed to develop ASD. IL-16, eotaxin, monocyte chemoattractant protein (MCP) 1, interferon (IFN) γ, IL-1β, IL-6 and IL-8 were unchanged in both groups at both 15 and 20 weeks. Conclusions: The current thesis has identified dysregulated expression of inflammation-driven molecular markers of the perinatal brain disorders HIE and ASD. Circulating levels of miRNAs (miR-128 miR-148a, miR-151a, miR-181a, miR-181b and miR-374a) and cytokines (IL-6, TNFα) were rapidly raised in response to HIE with and without inflammation-sensitisation in the porcine model. IL-17A was also dysregulated at 20 weeks’ gestation in mothers of ASD-affected children. These molecular markers may aid in rapid diagnosis, prognosis, and therapeutic decision-making for these time-sensitive disorders. Moreover, they may allow for discrimination of complex inflammation-sensitised HIE from classic HIE. Furthermore, evidence is beginning to suggest that HIE-associated miRNAs may play a functional role in the regulation of essential BMP-SMAD signalling. The current thesis puts forward novel information regarding the temporal profiles of these circulating molecular markers and begins to explore the functional roles of HIE-associated miRNAs. We hope this work will aid in the development of early blood-based biomarkers of disorders with an inflammatory milieu like HIE and ASD, and pave the way for more functional analysis of these markers.
Investigating metabolomic biomarkers of hypoxic ischaemic encephalopathy
(University College Cork, 2016) Denihan, Niamh Marie; Murray, Deirdre M.; Boylan, Geraldine B.; Kirwan, Jennifer A.; Molecular Medicine Ireland; Higher Education Authority
Background An early objective biomarker to predict the severity of hypoxic-ischaemic encephalopathy (HIE) and identify infants suitable for intervention remains elusive. This thesis aims to progress metabolomic markers of HIE through a pipeline of biomarker discovery and validation by employing a novel untargeted mass spectrometry metabolomic method. Methodology Term infants with perinatal asphyxia were recruited, all having umbilical cord blood (UCB) drawn and biobanked within three hours of birth. HIE was defined by Sarnat score at 24hours and continuous multichannel-EEG. Infant neurodevelopment was assessed at 36-42 months using the Bayley Scales of Infant and Toddler Development Ed. III (BSID-III). Untargeted metabolomic analysis of UCB was performed using direct injection FT-ICR mass spectrometry (DI FT-ICR MS). Putative metabolite annotations and lipid classes were assigned and pathway analysis was performed. Results Untargeted metabolomic analysis: Thirty enrolled infants were diagnosed with HIE, including 17 mild, 8 moderate, and 5 severe cases. Pathway analysis revealed that ΔHIE was associated with a 50% and 75% perturbation of tryptophan and pyrimidine metabolism respectively, alongside alterations in amino acid pathways. Significant metabolite alterations were detected from six putatively identified lipid classes including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids and prenol lipids. Outcome prediction: Metabolite model scores significantly correlated with outcome R=0.429 (model A) and R=0.549 (model B) respectively. Model B demonstrates the potential to predict both severe outcome (AUROC of 0.915) and intact survival (AUROC of 0.800). The effect of haemolysis: On average 5% of polar and 1.5% of non-polar features were altered between paired haemolysed and clean samples. However unsupervised multivariate analysis concluded that the preanalytical variability introduced by haemolysis was negligible compared with the inherent biological inter-individual variability. Conclusion This research has employed untargeted metabolomics to identify potential early cord blood biomarkers of HIE and has performed the technical validation of previously proposed markers.
Masks and tubes used to support the neonatal airway – how to improve their fit, seal and correct placement
(University College Cork, 2019-04-17) O'Shea, Joyce E.; Dempsey, Eugene M.; O'Connell, Liam; Davis, Peter; Thio, Marta; Kamlin, Omar
Despite the many changes in perinatal medicine in the last fifty years, infants still often and unpredictably need assistance with their breathing. Positive pressure delivered through a facemask remains the almost universal initial approach. This is then generally followed by endotracheal intubation if the infant is not responding or if prolonged support is needed. Despite many years of research into mask ventilation, it is still very challenging and leak and airway obstruction remain a problem. The thesis opens with two mask studies that try to solve this problem. The first is a manikin study that compared three different mask holds. It unfortunately found that there was no difference in the mask leak measured using the different holds. It is perhaps reasonable to change holds if the baby isn’t responding as expected. The second study aimed to measure the dimensions of preterm infants’ faces and compare these with the size of the most commonly available face masks. It found that the smallest size of some brands of mask is too large for many preterm infants. Masks of 35mm diameter are suitable for infants <29 weeks PMA or 1000g. Masks of 42 mm diameter are suitable for infants 27-33 weeks PMA or 750-2500g. The thesis then changed focus to neonatal intubation. Intubation is a challenging skill for paediatric trainees to master. In recent years success rates are decreasing. The next studies look at possible ways to change this trend. The first is a Cochrane review that examined if a stylet could improve intubation success. Only one unblended RCT has been performed and found no difference. The most sizable work of the thesis follows and is a RCT that examines if junior trainees intubation success rates are superior if they intubate with a videolaryngoscope. Two hundred and six intubations were randomised to the screen being visible to the supervisor or covered. The success rate when the instructor was able to view the videolaryngoscope screen was 66% (69/104) compared to 41% (42/102) when the screen was covered, (p<0.001), OR 2.81 (95%CI 1.54-5.17). This shows that videolaryngoscopy is a promising tool to help inexperienced trainees become proficient intubators. This study has resulted in videolaryngoscopy becoming a tool commonly used in neonatal intensive cares. The next study looks at recordings of unsuccessful intubations from the RCT. If an attempt is unsuccessful, the intubator and instructor often cannot explain why making it difficult to know what to do differently in the future. The study found that lack of intubation success was most commonly due to failure to recognize midline anatomical structures. Excessive secretions are rarely a factor in elective premeditated and routine suctioning should be discouraged. Better videolaryngoscope blade design may make it easier to direct the tube through the vocal cords. The final work of the thesis is a review that examines devices used during newborn stabilization. Evidence for their use to optimize the thermal, respiratory and cardiovascular management in the delivery room is presented. After completing all this work I think that perhaps it is time to lessen our reliance on facemasks and embrace other airway devices that are showing promise, particularly the laryngeal mask. I feel that universal intubation competency is no longer feasible but universal competency on the use of laryngeal masks probably is. This urgently needs to be addressed in paediatric training programs. Videolaryngoscopy is a promising tool that improves junior intubators’ success rates. To master intubation many intubations are still necessary but the videolaryngoscope allows the slope of the learning curve to steepen. Development is necessary to design scopes of the future that are inexpensive, easily portable and user friendly.
MicroRNA expression and function in neonatal hypoxic ischaemic encephalopathy
(University College Cork, 2016) Looney, Ann-Marie; Murray, Deirdre M.; Boylan, Geraldine B.; Cryan, John F.; Health Research Board
Hypoxic ischaemic encephalopathy (HIE) is a devastating neonatal condition which affects 2-3 per 1000 infants annually. The current gold standard of treatment - induced hypothermia, has the ability to reduce neonatal mortality and improve neonatal morbidity. However, to be effective it needs to be initiated within the therapeutic window which exists following initial insult until approximately 6 hours after birth. Current methods of assessment which are relied upon to identify infants with HIE are subjective and unreliable. To overcome this issue, an early and reliable biomarker of HIE severity must be identified. MicroRNA (miRNA) are a class of small non-coding RNA molecules which have potential as biomarkers of disease state and potential therapeutic targets. These tiny molecules can modulate gene expression by inhibiting translation of messenger RNA (mRNA) and as a result, can regulate protein synthesis. These miRNA are understood to be released into the circulation during cellular stress, where they are highly stable and relatively easy to quantify. Therefore, these miRNAs may be ideal candidates for biomarkers of HIE severity and may aid in directing the clinical management of these infants. By using both transcriptomic and proteomic approaches to analyse the expression of miRNAs and their potential targets in the umbilical cord blood, I have confirmed that infants with perinatal asphyxia and HIE have a significantly different UCB miRNA signature compared to UCB samples from healthy controls. Finally, I have identified and investigated 2 individual miRNAs; both of which show some potential as classifiers of HIE severity and predictors of long term outcome, particularly when coupled with their downstream targets. While this work will need to be validated and expanded in a new and larger cohort of infants, it suggests the potential of miRNA as biomarkers of neonatal pathological conditions such as HIE.
Multi-modal assessment of newborns at risk of neonatal hypoxic ischaemic encephalopathy – the MONItOr study
(University College Cork, 2022) Garvey, Aisling A.; Dempsey, Eugene M.; Murray, Deirdre M.; Boylan, Geraldine B.; National Children’s Research Centre, Crumlin, Ireland
Background: Hypoxic ischaemic encephalopathy (HIE) is the leading cause of acquired brain injury in term infants. At present, therapeutic hypothermia (TH) is the only approved therapy for infants with moderate-severe HIE. However, it must be commenced before 6 hours of age resulting in a clinical challenge to resuscitate, stabilize, identify and stratify infants in this narrow timeframe. Furthermore, a significant proportion of infants with mild HIE will have neurodevelopmental impairment. Improved, timely identification of infants at risk of brain injury is required. The aim of this study was to improve our knowledge of the early physiology of infants with HIE by describing the evolution of electroencephalography (EEG), near-infrared spectroscopy (NIRS) and non-invasive cardiac output monitoring (NICOM) in infants with all grades of HIE and to determine whether these markers may be helpful in the identification of infants at risk of brain injury. Methods: This prospective observational study was set in a tertiary neonatal unit (November 2017-March 2020). Infants with all grades of HIE had multi-modal monitoring, including EEG, NIRS and NICOM, commenced after delivery and continued for up to 84 hours. All infants had an MRI performed in the first week of life. Healthy term controls were recruited after delivery and had NICOM monitoring at 6 and 24 hours of age. In this thesis, I also included infants recruited previously as part of four historic prospective cohorts that had early EEG monitoring. These infants were combined with infants with mild HIE from the current prospective cohort to examine the difference in EEG features between infants with mild HIE and healthy term controls. Results: Eighty-two infants were recruited in the prospective cohort (30 mild HIE, 25 moderate, 6 severe, 21 controls) and 60 infants were included from the historic cohorts. This study identified significant differences between EEG features of infants with mild HIE and controls in the first 6 hours after birth. Seventy-two percent of infants with mild HIE had some abnormal features on their continuous EEG and quantitative analysis revealed significant differences in spectral shape between the groups. In our cohort, cSO2 increased and FTOE decreased over the first 24 hours in all grades of HIE regardless of TH status. Compared to the moderate group, infants with mild HIE had significantly higher cSO2 at 6 hours (p=0.003), 9 hours (p=0.009) and 12 hours (p=0.032) and lower FTOE at 6 hours (p=0.016) and 9 hours (0.029). Beyond 18 hours, no differences were seen between the groups. NICOM was assessed in infants with HIE and compared with controls. Infants with mild HIE have a significantly higher heart rate at 6 hours of age compared with controls (p=0.034). Infants with moderate HIE undergoing TH have a significantly lower cardiac output compared with mild HIE (p=0.046) and control groups (p=0.040). Heart rate is significantly reduced (p<0.001) but stroke volume is maintained and gradually increases from 6-72 hours despite TH. Finally, we assessed the ability of EEG, NIRS and NICOM to predict short-term outcome (abnormal MRI +/- death in the first week of life). At 6 hours, none of the EEG, NIRS or NICOM measures predicted short-term outcome. At 12 hours of age, both qualitative and quantitative EEG features significantly predicted abnormal short-term outcome. Conclusion: Identification of infants at risk of brain injury immediately after birth is challenging. Objective, early biomarkers are required. This is the first study to combine EEG, NIRS and NICOM in infants with all grades of HIE. Multi-modal monitoring is feasible and this thesis provides novel insights into the underlying physiology and evolution of injury in infants with HIE. Furthermore, it reaffirms the importance of early continuous EEG in HIE.
Neonatal resuscitation training in Ireland integrating teaching and research: a quality of care initiative in health services research
(University College Cork, 2001) Ryan, C. Anthony; Perry, Ivan J.
Resuscitation training is a vital area of education that could have a significant impact on patient outcome. This is especially true in the newborn period where inexperienced, inadequate or inappropriate resuscitation responses could affect the entire life span of a newborn infant. In addition, neonates are more often subject to asphyxia and are far more likely to be in need of resuscitation than any other age group. The World Health Organization (1995) estimates that every year there are one million neonatal deaths attributable to birth asphyxia. Due to the unique aetiology and pathophysiology of neonatal arrests, successful resuscitation requires the application of knowledge and skills that require special training. The purpose of resuscitation training is to transfer the science of resuscitation into classroom performance, with the objective of transferring knowledge and skills into clinical practice. The ultimate goal of the process is to improve neonatal survival and decrease handicap. While it is the responsibility of the Health Services to provide the necessary training for its employees, it is the function of Universities to design, develop, evaluate and promote effective educational programmes that improve patient care.
Neurodevelopmental outcome in perinatal asphyxia: prediction and measurement
(University College Cork, 2016) Ahearne, Caroline E.; Murray, Deirdre M.; Boylan, Geraldine B.; Health Research Board
The aim of this thesis was to improve our ability to predict and measure neurodevelopmental outcome in early childhood with particular reference to high-risk infants with perinatal asphyxia. Methods: 1) Promising umbilical cord blood biomarkers were analysed for ability to predict performance in the Bayley Scales of Infant and Toddler Development (Edition 3) at three years. 2) A retrospective cohort was analysed for performance of a low-risk cohort on the Bayley-3 at two years and compared to standardised scores. 3) A survey asked parents to report prevalence and quality of touch-screen usage in their toddlers. 4) Pilot testing of a novel cognitive assessment tool, the“Babyscreen App”, was performed on a prospective low risk cohort. The Babyscreen App was administered alongside the Bayley-3 at age 18 months to 2 years. Results: 1) IL-16 predicted severe outcome with an area under the ROC curve of 0.83 (p= Levels ≥ 514 pg/mL predicted a severe outcome with a sensitivity of 83% and a specificity of 81%. 2 metabolite models were tested. Model A predicted abnormal outcome with an area under ROC curve of 0.77, p<0.01. Model B was robust to predict both severe outcome (area under ROC curve of 0.92, p<0.01) and intact survival (0.80, p=0.01). 2) 240 two year olds were analysed for performance on the Bayley-3. Language and fine motor scores were significantly higher compared to U.S standardised norms, 109 ± 13 v. 100 ± 15 , p<0.001, and 11.5 ± 2 v. 10 ± 3, p<0.001 respectively. 3) For the examination of touch-screen usage in toddlers, 82 questionnaires were completed by parents of typically developing children aged 12 to 36 months. 71% of toddlers included had access to touch-screen devices for a median (IQR) of 15 (9-26) minutes per day. By 24 months the majority of children were able to swipe, unlock and actively look for touch-screen features. 4) 95 children underwent administration of the Babyscreen App and the Bayley-3. Significant medium sized correlations occurred between various measures of app performance and cognitive composite scores on the Bayley-3. Combined measures of overall app performance could predict cognitive scores less than 90 (1SD below the mean of our cohort) with an area under the ROC curve of 0.69 (0.55-0.83), p=0.02. Conclusion: This thesis has shown that novel biomarkers measured in umbilical cord blood at birth can predict neuro developmental outcome and that a novel touch-screen application can assess cognition in toddlers.
Neuromonitoring during newborn transition
(University College Cork, 2019) Finn, Daragh; Dempsey, Eugene M.; Boylan, Geraldine B.
Background: Newborn infant neurological function can be measured by monitoring electrical activity (electroencephalography) or cerebral oxygenation via NIRS (near infrared spectroscopy). In practice the clinical applications of electroencephalography (EEG) are limited to monitoring infants following moderate to severe hypoxic ischemic injury (HIE), and for the detection of seizures in at risk infants. NIRS monitoring has been the focus of a number of research trials but has no clinical applications in the immediate newborn period to date, and is not routinely performed in neonatal units. Aim: To assess the feasibility of infant neuromonitoring in the immediate period in two important clinical scenarios. Firstly, to assess the feasibility of monitoring brain activity during the first minutes of life in healthy term infants. Secondly, to assess the feasibility and utility of monitoring newborn preterm infants’ brain activity and cerebral oxygenation in the context of an interventional randomized controlled trial. Methods: 1. Healthy term newborn infants had EEG monitoring performed for the first ten minutes of life. EEG was assessed both qualitatively and quantitatively. All infants had respiratory function monitoring performed simultaneously. 2. Forty-five infants (< 32 weeks gestation) were randomly assigned to different methods of newborn infant cord clamping. All infants had EEG and NIRS monitoring for the first 72 hours of life. Quantitative features of EEG and median NIRS values were compared between groups at 6 and 12 hours of life as a primary outcome measure. Results: 1. Forty-nine infants had EEG recordings. Median (IQR) age at time of initial EEG recording was 3.0 (2·5 to 3·8) minutes. End tidal CO2 and tidal volumes increased over the first 3 minutes of life and then stabilized. Good quality EEG, with continuous mixed frequency activity with a range of 25-50μV, was observed in all infants. The majority of EEG spectral power was within the delta band. 2. There were 45 infants included. One infant died in the delivery room. Median time (IQR) from birth until EEG application was 3.05 (1.85 to 5.38) hrs. For primary outcome measures, data was available for 42/44 (95%) at 6 hrs and 44/44 (100%) at 12 hours. There was no significant difference between groups for measures for EEG values or cerebral NIRS. Conclusion: Infant neuromonitoring in the immediate newborn period is feasible in the first minutes of life in healthy term infants and within the first hours of life in preterm infants. Normative quantitative data for electrical activity in healthy newborn term infants during the first minutes of life is described for the first time. Neuromonitoring during the first day of life as an outcome measure for preterm interventional trials is possible and the outcomes from this research is promising for further trials.
Refining the evaluation of growth and the growth hormone/insulin-like growth factor-I axis in children
(University College Cork, 2018) Hawkes, Colin P.; Murray, Deirdre M.; The National Children's Research Centre
Introduction: The growth hormone (GH)/Insulin-like growth factor-I (IGF) axis is a key mediator of childhood growth. Current diagnostic tests have poor specificity for disorders affecting this system, namely the growth hormone stimulation test (GHST) and IGF-I measurement. Aim: To improve the diagnostic evaluation of children with poor growth and possible GH deficiency (GHD) through 1) modifying the GHST and diagnostic fasting study; 2) utilising liquid chromatography mass spectrometry (LCMS) to measure IGF concentrations; 3) exploring genetic causes of poor growth; and 4) studying the association between body composition and infant growth. Methods: These include: additional GH measurements during the GHST and fasting study; IGF-I and –II measurement by LCMS in a well-characterised cohort; focused whole exome testing for rare clinical phenotypes; and body composition analysis using air displacement plethysmography. Results: Serial additional GH measurement after intravenous catheter placement will improve the specificity of the GHST. Similarly, serial GH measurement after a diagnostic fasting study will improve specificity for GHD. In normal infants, adiposity doubles in the first two months. Using LCMS, I have described reference data for IGF-I and –II at birth and demonstrated a relationship between these measurements and this rapid early accumulation of body fat. In our genetic studies, we have also identified a novel IGF1R mutation in a child with a phenotype consistent with IGF-I resistance. Conclusions: Diagnosing disorders of the GH/IGF-I axis remain a significant clinical challenge. I have expanded the clinical approach to evaluating the child with short stature through refining the GHST, diagnostic fasting study and body composition evaluation; describing reference data for body composition and IGFs in infancy; and exploring novel genetic causes of disordered growth. Future work will focus on studying other clinical tools in evaluating the child with short stature and predicting the clinical response to GH treatment.

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