Paediatrics & Child Health - Doctoral Theses

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    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.
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    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.
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    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.
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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.
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    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.