Neonatal Society Autumn Meeting 2012

The Institute of Child Health, London

Thursday 8th November

Autumn Meeting, Thursday 8th November 2012 Institute of Child Health, Guilford Street, London, WC1N 1EH. 09.30 Coffee Session 1: Chair – Dr Richard Thwaites 09.45 S Broster, Acute Neonatal Transfer Service for the East of England Active versus passive cooling during neonatal transfer 10.00 K Butler, School of Medicine, University of Cardiff and University Hospital Wales

Is that blood test really necessary? A prospective, observational study conducted in a tertiary Neonatal Intensive Care Unit

10.15 S Grossman, Homerton University Hospitals NHS Foundation Trust, London Can Umbilical Cord Bilirubin be used to Predict Neonatal Jaundice in Term Infants? 10.30 L Burgess, Department of Neonatology, Liverpool Women’s Hospital

Plasma arginine levels and blood glucose control in very preterm infants receiving two different parenteral nutrition regimens

10.45 M Johnson, National Institute for Health Research, Southampton Biomedical Research Centre

Early parenteral nutrition and growth outcomes in preterm infants: a systematic review and meta-analysis

11.00 Neonatal Society Student Prize: Simon Biddie, University of Bristol Genome-scale discovery of developmental regulatory elements 11.15 Morning Coffee Session 2: Chair – Dr James Boardman 11.45 J Parkinson, Section of Neonatal Medicine, Chelsea and Westminster Campus, Imperial

College London Urinary metabolome in ex-preterm adults following postnatal dietary intervention 12.00 P Fleming, Blizard Institute of Cell and Molecular Science, Barts and the London School of

Medicine and Dentistry Bacterial Translocation preceding necrotising enterocolitis and sepsis in infants below 31 weeks gestational age

12.15 N Andreas, Section of Neonatal Medicine, Chelsea and Westminster Campus, Imperial College

London Variation in Breast Milk Composition over Time using 1H Nuclear Magnetic Resonance Spectroscopy

12.30 Annual General Meeting for Members of the Neonatal Society 13.15 Lunch Break

Session 3: Chair – Dr Helen Budge 14.15 D Gibbons, Department of Immunobiology, Kings College London, Guys Hospital Human neonatal CD4 T cells display distinct functional potentials relative to adults 14.30 M Boyle, Neonatal Department, The Rotunda Hospital, Parnell Square, Dublin 1, Ireland Expansion of Ventricular Indices to Include Extremes of Prematurity 14.45 S Pauliah, Institute for Women’s Health, University College London

Therapeutic Hypothermia for Neonatal Encephalopathy in Low- and Middle-income Countries: A Systematic Review and Meta-analysis

15.00 Keynote Lecture: Dr Simon Waddington, University College London ‘Perinatal Gene Therapy’. 15.45 Afternoon Tea Session 4: Chair – Dr Matthew Hyde 16.15 I Tachtsidis, Department of Medical Physics and Bioengineering, University College London

Investigation of brain tissue oxygenation, cytochrome-c-oxidase measured with NIRS and intracellular metabolites measured with MRS during perinatal cerebral hypoxia-ischaemia

16.30 S Jary, Neonatal Neuroscience, School of Clinical Science, University of Bristol

Comparison of Bayley-2 and Bayley-3 Cognitive and Motor outcome at 18 months in infants with Neonatal Encephalopathy treated with Hypothermia.

16.45 H Sabir, School of Clinical Sciences, University of Bristol, St Michael’s Hospital, Bristol

Neither Xenon nor Fentanyl Induce Neuroapoptosis in the Newborn Pig Brain, however Isoflurane does.

17.00 F Moultrie, University of Oxford Characterising the early development of human pain processing and behaviour 17.15 The Widdowson Lecture: Professor Michael Weindling, University of Liverpool, Introduced by Professor Neena Modi, President of the Neonatal Society 'Tissue oxygenation in the newborn infant' 18.15 Drinks and Close of Meeting

Title: Active versus passive cooling during neonatal transfer

Authors: Chaudhary R1,Broster S

1, Farrer K

1, Austin T

2

Institution:1Acute Neonatal Transfer Service for the East of England,

2Neonatal Intensive Care Unit, Rosie

Hospital, Cambridge University Hospitals NHS Foundation Trust, UK.

Background/ Introduction Therapeutic hypothermia is now recommended as the standard of care for the management of infants with moderate or severe hypoxic-ischaemic encephalopathy (HIE)

1. Current practice in the UK is that most infants

are treated in regional neonatal intensive care units (NICUs). Evidence suggests treatment should begin as soon as possible after birth thus cooling should be initiated at the referring hospital and continued during transfer to the regional NICU. A number of small series have reported the experience of passive and active cooling during transfer of these infants, but to date there is no published large comparative series or recommended consensus on the optimal method for maintaining hypothermia during transfer

2,3.

Methods The Acute Neonatal Transfer Service (ANTS) for the East of England have delivered therapeutic hypothermia during transfer since June 2009. In October 2009 the service hours increased to 24 hours daily. Initially infants were stabilised and transferred using passive cooling methods. Since March 2011 infants have been actively cooled using a servo-controlled mattress. The aim of this retrospective observational study was to compare the effectiveness of passive and active cooling during transfer. The key outcome measurements were: time to target temperature, stabilisation time and temperature stability during transfer. Target temperature range was defined as 33.0-34.0

oC. As a marker

of temperature stability the percentage of time within this range during transfer was calculated for each infant. Unpaired t-test was used to compare the differences between the two different groups. Results Between June 2009 and May 2012 a total of 143 infants with HIE requiring cooling were referred for transfer and 134 transferred. Passive cooling methods were used in all infants by the referring hospital to initiate therapeutic hypothermia. Following arrival of the ANTS team, cooling was maintained passively in 64 infants until arrival at the regional NICU and 70 were actively cooled using the servo-controlled Tecotherm Neo (Inspiration Healthcare). There was no significant difference in gestation, birthweight, sex, or age at referral between the two groups. The mean (+/-SD) time cooling was initiated in the passive group was 141 (+/-121) minutes and 80 (+/-85) minutes in the active group (t=3.31, p=0.001). The mean (+/-SD) stabilisation time in the passive group was 178 (+/-81) minutes and 151 (+/-77) minutes in the active group (t=2.03, p=0.04). The mean (+/-SD) time within target temperature range in the passive group was 45.8 (+/-35.8)% and 81.2 (+/-21.5)% (t=6.53, p=0.0001). 34.4% of infants transferred passively cooled were overcooled (temperature <33

oC) compated with 11.2% of infants actively cooled.

Conclusions These data suggest that while initiation of therapeutic hypothermia is achievable with passive methods, maintenance and stability of temperature is significantly enhanced using a servo-controlled cooling mattress. Additionally the thermal stability of the active method results in a significant reduction in stabilisation time and allows the transfer team to focus on other aspects of the infants’ management. Furthermore active cooling during the journey prevents potentially serious over-cooling. Servo-controlled cooling should be considered by all transfer teams as a safe and effective way of managing infants with HIE during transfer.

References 1. NICE IPG374: Therapeutic hypothermia with intracorporeal temperature monitoring for hypoxic perinatal brain

injury: guidance. Issued May2010 http://www.nice.org.uk/nicemedia/live/11315/48809/48809.pdf 2. Kendall GS, Kapetanakis A, Ratnavel N, et al. Passive cooling for initiation of therapeutic hypothermia in neonatal encephalopathy.

Arch Dis Child Fetal Neonatal Ed. 2010;95:F408-12. 3. O’Reilly K, Tooley J, Winterbottom S. Therapeutic hypothermia during neonatal transport. ActaPaediatrica 2011;100:1084-6.

Corresponding Author email address:topun.austin@nhs.net Check box to confirm if Presenting Author is a trainee Senior Author supporting presentation on the day of the meeting: This abstract has not been published elsewhere

Title: Is that blood test really necessary? A prospective, observational study conducted in a tertiary Neonatal Intensive Care Unit

Authors

Katherine Butler, Cora Doherty

Institution:

School of Medicine, University of Cardiff & University Hospital Wales, Cardiff

Background/ Introduction

There has been a recent drive for efficient working practice to allow more time for direct clinical care and reduce unnecessary expenditure within the NHS

1. The European working time directive (EWTD) and the balance of service

versus training for junior doctors are further drivers for efficiency. Furthermore, with a potential reduction in medical trainees it is important not only to ascertain transferable skills but to identify unnecessary tasks, particularly if they impact on the patient. Within intensive care many blood tests are labelled ‘routine’ and thought necessary however pathology services have identified a 20% increase in tests over the last decade

2. We have previously calculated that approximately

50 hours per week are spent performing blood tests within our tertiary neonatal unit3. Our aim was to determine which

indications for blood tests were associated with a clinical intervention post results and to determine whether ‘unnecessary’ blood tests could be identified.

Methods

A prospective, observational study was performed over 120 hours on a tertiary NICU in two rooms which had level 3 babies. Each blood test performed was recorded including information on the indication for testing and any clinical intervention following test results. To help standardise the blood test ‘indications’, a checklist was developed. Minute-by-minute process mapping for each blood test was also recorded. The 120 hours consisted of five 12 hour night shifts (9pm-9am) and five 12 hour day shifts (9am-9pm). Verbal consent was obtained from all NICU staff on duty prior to the start of each ‘study’ shift.

Results

285 blood tests were taken over 120 hours accounting for a total of 2147 minutes (30% of total time). There were 30 (10.5%) ‘failed’ or ‘lost’ samples. 154 (54%) tests led to an action changing patient management. Clinical Interventions were then related to the test indicator. ‘Monitoring ventilation’, ‘monitoring trends’ and ‘Change in ventilation type/settings’ were the three commonest indicators identified with interventions made in 41%, 73%, and 83% of test results respectively. 101 (35.5%) resulted in no clinical intervention.

Type of Blood Test successfully

performed (% of total)

Number Resulting in an

intervention

No intervention

Blood Gas 154 (54%) 80 (52%) 60 (39%)

FBC 41 (14%) 24 (59%) 11 (27%)

U&E 35 (12%) 15 (43%) 16 (46%)

Therapeutic drug levels 12 (4%) 12 (100%) 0 (0%)

Conclusions

Within a tertiary NICU 54% of blood tests on level 3 babies resulted in a clinical intervention post result. However 35.5% blood tests resulted in no intervention. Whilst doing some apparent ‘unnecessary’ tests may be unavoidable, more consideration when deciding whether or not a test is performed could reduce laboratory costs and free up time for training. More importantly however neonatal pain and blood loss could be minimised.

References

1. http://www.wales.nhs.uk/sites3/page.cfm?orgId=781&pid=31388 sited 11/11/2011

2. Rao, G., et al. Pathology tests: is the time for demand management ripe at last? Journal of clinical Pathology 2003; 56(4): 243–248

3. Taylor, A., Doherty, C. 5000 minutes in NICU - An observational study of doctors’ time on a tertiary NNU as part of the Transforming Care Initiative, presented Neonatal Society, March 2012

Corresponding Author email address: Butlerk1@cardiff.ac.uk Check box to confirm if Presenting Author is a trainee: Final Year Medical Student Senior Author supporting presentation on the day of the meeting: Dr Cora Doherty This abstract has not been published elsewhere

Title: Can Umbilical Cord Bilirubin be used to Predict Neonatal Jaundice in Term Infants?

Authors: Sophie Grossman, 1,2

Kelsey D J Jones,1 Dharshini Kumaranayakam,

1,2 Arati Rao,

1 Greg Fegan,

3

Narendra Aladangady.1,2

Institutions: 1. Homerton University Hospitals NHS Foundation Trust, London, UK 2. Barts and the London School of Medicine & Dentistry, Queen Mary, University of London 3. Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford Background: Neonatal jaundice is well recognised as one of the commonest causes of admission to hospital in the neonatal period amongst term babies in all settings, and is associated with substantial morbidity

1-3. Although with prompt and effective management the incidence of Kernicterus is very low

4, the

devastating nature of this condition coupled with the substantial economic burden of jaundice-related hospitalisations means that strategies to improve early recognition of clinically significant hyperbilirubinaemia remain a public health priority.

Objectives: To assess whether arterial umbilical cord bilirubin (aUCB) level at delivery predicts the development of clinically significant neonatal jaundice in term infants. Methods: Retrospective analysis of hospital biochemistry records between February – November 2010

identified term deliveries with recorded aUCB. Infant medical records were reviewed to identify those who

developed clinically significant neonatal hyperbilirubinaemia with/without a positive direct antiglobulin test

(DAT), or sepsis according to clinical criteria. The study was approved by the UK National Research Ethics

Service (NRES) Committee London – Harrow (reference 11/LO/0796).

Results: Of 1411 term deliveries with a clearly recorded aUCB, 30 infants developed clinically-significant jaundice (2.7%), of whom 8 were DAT+ve (0.6%) mostly due to ABO incompatibility, and 42 developed clinically-defined sepsis. aUCB strongly predicted the development of DAT+ve jaundice (area under the ROC curve = 0.996), as well as all-cause jaundice (area under the ROC curve = 0.74). However, this effect was critically dependent on maternal blood group. Amongst infants at risk of ABO incompatibility (maternal blood groups O+ve/O-ve, 43.8%) the predictive value of aUCB for all cause jaundice was strengthened (area under the ROC curve = 0.88). Amongst those not at risk (maternal blood group not O+ve/O-ve, 53.1%) it disappeared completely (area under the ROC curve = 0.46). aUCB had no predictive value for sepsis. Conclusions: For infants of mothers with blood group O, aUCB predicts development of neonatal jaundice. There was no evident utility for infants of mothers with different blood groups. Estimation of aUCB should be considered as a strategy for early identification of those at risk of neonatal haemolytic jaundice.

References 1. Sarici S U et al. Pediatrics 2004;113:775–780.

2. Escobar GJ et al. Arch Dis Child. 2005;90(2):125-131.

3. Mwaniki MK et al. BMC Public Health. 2010;10:591.

4. Johnson L et al. J Perinatol. 2009;29:S25-45. Corresponding Author email address: Sophie.grossman8@gmail.com, narendra.aladangady@homerton.nhs.uk

Check box to confirm if Presenting Author is a trainee: clinical trainee √ basic science trainee

Senior Author supporting presentation on the day of the meeting: Dr Narendra Aladangady

This abstract has not been published elsewhere

Title: Plasma arginine levels and blood glucose control in very preterm infants receiving two different parenteral nutrition regimens

Authors: Burgess L1, Morgan C

1, Mayes K

2, Tan M

2

Institution: 1Department of Neonatology, Liverpool Women’s Hospital;

2Alder Hey Children’s Hospital

Background/ Introduction: We have previously shown1 that improving early protein intake is associated with a

reduction in insulin-treated hyperglycaemia in preterm infants <29 weeks gestation1. While the exact

mechanism for this effect is unknown, the affect of amino acids (AA) on insulin secretion is well described in preterm infants. Some AAs are more potent secretogoges than others. The arginine stimulation test is routinely used in clinical adult practice to assess insulin secretion by the pancreas. Similar neonatal effects have been demonstrated but the implications of arginine intake for the hyperglycaemic preterm infant has not been studied. We hypothesised that low arginine levels would be associated with an increase in insulin treated hyperglycaemia and higher mean daily blood glucose levels (day1-15) in infants born <29 weeks gestation. Methods: We used the methods described in our previous study

1 to perform a secondary analysis on the data

from a previous randomised controlled trial comparing a hyperalimentation and a standard neonatal PN

regimen (control).2 The hyperalimentation regimen was designed to provide 20% more carbohydrate than the

control regimen thereby providing a greater test of glucose tolerance for these infants. Daily carbohydrate and

protein intake data and mean daily blood glucose and insulin use data from the first 15 days of life were

substratified according to high arginine (highARG) or low arginine (lowARG) levels on day 8-10. The threshold

for high/low arginine was 57micromol/l (based on the median plasma level in a separate reference population).

Results: Of the 60 control group infants (C) with day 8-10 arginine data, 41 were substratified to the lowARG

group and 19 to the highARG group. There were no differences in basic demographic factors likely to affect

blood glucose. There were no differences in carbohydrate or protein intake. The blood glucose data replicated

our previous findings showing a peak in hyperglycaemia on day 5-10. Low arginine levels were associated

higher mean daily blood glucose levels (day 6-10) and more insulin treatment (Table 1; group C). Of the 60

hyperalimentation group infants (H) with day 8-10 arginine data, 33 were substratified to the lowARG group and

22 to the highARG group. LowARG infants were of lower gestation and birthweight (p<0.01) There were no

differences in carbohydrate or protein intake. Low arginine levels were associated higher mean daily blood

glucose levels (day 1-5, 6-10) and more insulin treatment (Table 1; group H).

Table 1: Mean (SE) blood glucose (mmol/l; 5 day time periods) and insulin use (total days, d1-15)

Group d1-5 (C) d6-10 (C) d11-15 (C) Insulin d1-5 (H) d6-10 (H) d11-15 (H) Insulin

lowARG

highARG

p value

6.9 (0.3)

6.6 (0.6)

0.58

8.6 (0.4)

7.3 (0.4)

<0.05

7.1 (0.4)

6.0 (0.5)

0.11

110

30

8.2 (0.4)

6.7 (0.4)

<0.01

9.9 (0.4)

8.3 (0.6)

0.02

7.6 (0.4)

6.6 (0.4)

0.11

203

66

Conclusion: Low plasma arginine levels in very preterm infants are associated with poorer blood glucose

control (measured by mean daily blood glucose and insulin treatment). Further RCT evidence is required.

References: 1. Mahaveer A, Grime C, Morgan C. Nutr Clin Pract;27:399-405. 2. Tan M, Cooke R. Arch Dis Child Fetal Neonatal Ed 2008;93:F337-341 Corresponding Author email address: colin.morgan@lwh.nhs.uk

Presenting author: Laura Burgess supported by Consultant Neonatologist: Colin Morgan

This abstract has not been published elsewhere

Title: Early parenteral nutrition and growth outcomes in preterm infants: a systematic review and meta-analysis

Authors (Presenting author underlined. If no author is a Society member please provide the name of the member introducing the author to the Society) Mark J. Johnson

1,2, Helen E. Moyses

1, Alison A. Leaf

1,2, Victoria R. Cornelius

3

Institution: 1.National Institute for Health Research, Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton 2. Department of Neonatal Medicine, University Hospital Southampton NHS Foundation Trust, Southampton 3. Department of Primary Care and Public Health Sciences, Kings College London

Background/ Introduction (to include Hypothesis and Ethics Approval if required) Achieving adequate nutritional intakes in preterm infants is challenging, and may explain poor growth often seen in this group. The use of early parenteral nutrition (PN) has been proposed as one way of addressing this issue, although the benefit and harms are unknown. We carried out a systematic review to assess the effect of timing of commencement of PN on growth and risk of morbidity and mortality in preterm infants.

Methods The databases MEDLINE, EMBASE, CINAHL, 'Web of Science’ and 'CSA-Conference Papers Index' were searched between 1947 and July 2012, with selective citation and reference searching. Studies were eligible if they were RCTs or observational studies comparing ‘early’ versus ‘late’ PN or intravenous amino acids in preterm infants, and had at least one growth outcome. The definition of ‘early’ was within 48 hours of birth, and studies where the ‘early’ feeding intervention was later than 48 hours were excluded. There was no restriction on language.

Results (may contain tables and figures) Eight RCTs and 13 observational studies met inclusion criteria (n=553 and 1796 infants). Meta-analysis was limited by disparate growth outcome measures. Assessment of potential bias was difficult to gauge due to inadequate reporting. Results are given as mean difference (95% Confidence Interval). Early PN reduced time to regain birth weight by 2.2 days (1.1-3.2) for RCTs and 3.2 days (2.0-4.4) in observational studies. Maximum percentage weight loss was lower with early PN by 3.1% (1.7-4.5) for RCTs and 3.5% (2.6-4.3) for observational studies. Early PN improved weight at discharge/36 weeks by 14.9g (5.3-24.5, observational studies only). No benefit was found for length or head circumference at discharge/36 weeks. There was no significant difference in the risk of mortality, necrotising enterocolitis, sepsis, chronic lung disease, VH or cholestasis between the early and late groups.

Conclusions The results of this review whilst subject to some limitations, demonstrates that the use of early PN provides benefit for short term growth outcomes, with some evidence of benefit for growth outcomes at discharge or 36 weeks. No evidence that early PN causes harm was found. Growth measures in neonatal research would benefit from the development of a set of core outcome measures. References (key references should be included) 1. Embleton NE, Pang N, Cooke RJ. Postnatal malnutrition and growth retardation: an inevitable

consequence of current recommendations in preterm infants? Pediatrics 2001;107(2):270 2. Ehrenkranz RA. Early, aggressive nutritional management for very low birth weight infants: what is the

evidence? Semin Perinatol 2007;31(2):48-55. Corresponding Author email address: m.johnson@soton.ac.uk

Check box to confirm if Presenting Author is a trainee: clinical trainee basic science trainee

Senior Author supporting presentation on the day of the meeting: Dr Alison Leaf

This abstract has not been published elsewhere

Title: Genome-scale discovery of developmental regulatory elements

Simon C Biddie 1,2,3

, Catia Attanasio 2, Alex S Nord

2, Matthew J. Blow

3, Axel Visel

2,3 and Len A.

Pennacchio 2,3

1. Faculty of Medicine and Dentistry, University of Bristol, Bristol, UK 2. Genomics Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 3. United States Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA.

Background Mutations in coding regions of the human genome are well known to cause disease, for example exonic mutations causing cystic fibrosis and MCADD. However, genome-wide association (GWA) studies have identified extensive genetic polymorphisms in non-coding elements that confer disease risk. The non-coding DNA sequences represent ~98% of the human genome, and harbour regulatory elements such as enhancers that act to regulate gene expression. Enhancers regulate genes by the binding of regulatory factors, co-factors and through the modification of histones, the DNA compacting protein complexes. Mutations in regulatory elements could therefore alter gene expression in disease. The identification of enhancers across the genome has previously been challenging as their activities differ between tissues and change during development. However, the recent emergence of whole-genome sequencing technologies has aided enhancer identification. Mapping of an enhancer-bound co-activator protein, p300, has been successfully used in unbiased discovery of tissue-specific enhancers active in the developing mouse embryo and human foetal heart (1,2). However, p300 is associated only with a subset of enhancers, leaving an unknown number of undiscovered enhancers. Regulatory elements can also act as repressors that silence gene expression, a class of genomic elements that remains poorly investigated. New methods and markers of enhancer and repressors would expand our understanding of non-coding elements that might be associated with disease.

Methods Active regulatory elements were identified by chromatin immunoprecipitation with high-throughput sequencing (ChIP-Seq) of the enhancer-associated chromatin remodelling subunit Brg1 (SMARCA4) in E11.5 mouse heart, forebrain and limb bud. Due to limited availability of antibodies against Brg1, FLAG-tagged Brg1 knock-in mice were generated, with anti-Flag antibodies used for immunoprecipitation. Putative enhancer elements were validated in vivo using a LacZ transgenic mouse assay.

Results Brg1 binding sites were found to be highly tissue-specific, consistent with the possibility that they are involved in tissue-specific gene regulation. A subset of Brg1 binding sites were found to be associated with enhancer-associated chromatin marks including H3K27ac. A substantial proportion of predicted Brg1-associated enhancer elements showed reproducible tissue-specific enhancer activity in vivo in a transgenic mouse assay. Furthermore we observed a proportion of Brg1 peaks overlapping tissue-specific binding sites for repressor-associated chromatin signatures across the genome, suggesting that Brg1 is also commonly associated with repressors. Incorporating GWA data with Brg1 at human and mouse conserved DNA elements, we found elements harbouring single nucleotide polymorphisms associated with disease.

Conclusions Whole-genome mapping of Brg1 occupancy in developing mouse tissues identified genome-wide sets of candidate regulatory sequences that are likely to be involved in tissue-specific transcriptional regulation in embryo development. Comparison with tissue-specific histone marks, as well as in vivo enhancer assays in transgenic mice, indicate that Brg1 is widely associated with both tissue-specific enhancers and repressor elements. Our results provide genome-scale evidence for a dual role of this key regulatory protein in both tissue-specific activation and repression of developmentally expressed genes. These elements identified through this study provide candidate sequences in the search for non-coding sequence polymorphisms that might underlie human congenital disease.

References 1. Visel, A et al.,2009.ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature, 457(7231) 2. May, D et al.,2011.Large-scale discovery of enhancers from human heart tissue. Nature Genetics, 44(1)

Corresponding Author: Simon.Biddie@doctors.org.uk East of England Foundation School, Cambridge University Hospitals NHS trust, Cambridge, UK

Urinary metabolome in ex-preterm adults following postnatal dietary intervention

Parkinson JRC1, Hyde MJ

1, Wijeyesekera D

2, Holmes E

2, Singhal A

3, Modi N

1

1 Section of Neonatal Medicine, Chelsea and Westminster campus, Imperial College London

2 Section of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London

3 Clinical Trials & Cardiovascular Nutrition Group, MRC Childhood Nutrition Research Centre, ICH, London

Background/Introduction

Optimising nutrition represents a key therapeutic window for improving outcomes for preterm babies in both infancy and

adulthood. We have previously presented data to the Society showing preterm adults demonstrate an altered urinary

metabolic profile compared to term born controls; including increased acetylated glycoprotein fragments, associated with

inflammation and reduced hippurate, which has an inverse correlation with blood pressure (1). The aim of this study was

to determine if postnatal nutritional intervention alters metabonomic profile in ex-preterm adults.

Methods

We analysed urine samples collected during a follow-up of a cohort of ex-preterm adults (aged 19-20 years, gestational

age (GA) (mean ± SD: 30.9 ± 2.7 years)) randomised to banked breastmilk (BBM), term formula (TF) or preterm formula

(PTF) for an average of one month postnatally (2). 1H NMR spectroscopy was performed on urinary samples. Spectra

were analysed by dietary group and by gender. Orthogonal projection to latent structure discriminant analyses (O-PLS-

DA) was used to model class differences, and identify metabolites contributing to the differences between groups.

Gender specific group comparisons were calculated for (1) higher nutrient (PTF) versus lower nutrient (TF and BBM

combined), (2) PTF vs. BBM groups and (3) PTF versus TF. In addition, spectra were correlated with birth weight (bwt),

GA and 2 week bwt z scores.

Results

We studied 197 healthy young adults (BBM, n=55 (28 men)), term formula (TF: n=48 (14M)) or preterm formula (PTF,

n=94 (40M)). We found no significant differences in urinary spectra between dietary groups when combined or analysed

in a gender specific fashion. Correlation analysis revealed a significant correlation between GA and preterm spectra (R2:

0.65, Q2: 0.26). Metabolites significantly correlated with increased GA are listed in Table 1.

Metabolite ppm Increase/Decrease with gestational age

N-Acetyl-5-hydroxytryptamine 1.91 +

3-Methyl-L-histidine 3.69 +

Hippurate 3.69 +

4-cresyl sulphate 7.2 +

Table 1. Metabolites significantly associated with gestational age

Conclusions

Despite a number of physiological benefits observed in ex-preterm individuals randomised to BBM (2), we observed

no differences in metabolic profile between dietary intervention groups. In line with previous metabonomic data an

increased GA was associated with increased hippurate (1) and 4-cresyl sulphate, metabolites associated with

reduced blood pressure, improved gut microflora and lower BMI (3). In addition to the established inverse correlation

between prematurity and blood pressure, these data suggest that gut health in ex-preterm adults is also dependent

on the degree of prematurity.

References

(1) Aberrant adiposity and ectopic lipid deposition characterize the adult phenotype of the preterm infant. Thomas et

al. Pediatr Res. (2011) Nov;70(5):507-12.

(2) Early diet and peak bone mass: 20 year follow-up of a randomized trial of early diet in infants born preterm.

Fewtrell et al. (2009) Bone 45 142–149

(3) Quantitative UPLC-MS/MS analysis of the gut microbial co-metabolites phenylacetylglutamine, 4-cresyl sulphate

and hippurate in human urine. Wijeyesekera A et al. (2012) Anal. Dyn Meth 2012, 4, 65

We acknowledge assistance from: Maria Kokoreli and Adam Lewandowski

Corresponding Author email address:jrcp@imperial.ac.uk

Check box to confirm if Presenting Author is a trainee: clinical trainee

basic science trainee

Senior Author supporting presentation on the day of the meeting: Prof Neena Modi

This abstract has not been published elsewhere

Title: Bacterial Translocation preceding necrotising enterocolitis and sepsis in infants

below 31 weeks gestational age

Authors:PF Fleming, N Panton, M Wilks, M Millar, KL Costeloe Institution: Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Whitechapel E12AT; Homerton University Hospital, Homerton Row, E96SR, Royal London Hospital, Whitechapel E1 1BB.

Background/ Introduction

Loss of the protective barrier function of the intestinal wall with associated invasion and perhaps

translocation by bacteria, are regularly cited to be involved in the pathogenesis of necrotising enterocolitis

(NEC) and septicaemias in preterm infants(1)

.In the case of NEC, whether such bacterial invasion occurs in

association with the inflammation that characterises the clinical onset of the disease, or whether it may

precede symptoms is not known. In vivo studies investigating this concept are limited and constrained

because of paucity of data preceding the onset of symptoms. Hypothesis: That bacterial translocation of

the intestinal wall may occur in asymptomatic preterm infants and precede the clinical onset of NEC.

Methods After informed written consent (HREC 10/H0802/40), infants <31 weeks of gestation, were enrolled during the second week into a study of intestinal permeability. Circulating bacterial material was detected using 16S rDNA. Blood samples were collected weekly starting at 14 days (+/- 2 days) for 4 weeks and frozen at -80˚C within 4 hours. 16S detection was performed using a probe-based real-time PCR assay (Nadkarni et al 2002). Suitable controls were included in each run. Clinical outcomes were recorded.

Results

213 samples were collected from 61 infants (33 male and 28 female), mean (SD) birth weight 913g (213)

and median (range) gestation 26 weeks (23-30). 8 infants (13%) developed NEC (≥Bells Stage II) at a

median age of 26 days (range 19-72), two infants died from NEC related complications.

11/213 samples (5%) were positive for bacterial 16S rDNA. Six positive samples were collected from 3 infants who subsequently developed stage 3 NEC. The 5 remaining positive PCRs were from 5 infants none of whom developed NEC but 3 of whom had clinical sepsis (1 Klebsiella , 1 CONS septicaemia and 1 cellulitis) within 24-48 hours of sampling.

Conclusions

We have demonstrated circulating bacterial material in the blood of asymptomatic preterm babies 6 out of 8

of whom went on to become ‘septic’ or to develop NEC. We speculate that in babies who developed NEC,

this material entered the blood stream by translocation of the intestinal wall.

References: Sherman MP. New concepts of microbial translocation in the neonatal intestine: mechanisms and prevention. Clin Perinatol. 2010 Sep;37(3):565-79.

Corresponding Author: drpaulfleming@gmail.com

Check box to confirm if Presenting Author is a trainee: clinical trainee

basic science trainee

Senior Author supporting presentation on the day of the meeting: KL Costeloe

This abstract has not been published elsewhere

Title: Variation in Breast Milk Composition over Time using 1H Nuclear Magnetic Resonance Spectroscopy

Authors: Nicholas J. Andreas, Isabel Garcia-Perez, Suzan Jeffries, Chris Gale, Matthew J. Hyde, Elaine

Holmes and Neena Modi Institution: Section of Neonatal Medicine, Chelsea & Westminster Campus, Imperial College London, UK

Background/Introduction: Breast milk composition is known to vary widely, both temporally (each day, and throughout lactation) and between individual mothers. How maternal characteristics determine breast milk composition is poorly understood. We are currently using a range of high throughput technologies to extract maximal compositional data on breast milk in order to study the variations in milk micronutrient content.

1H NMR provides a robust and repeatable way of determining the metabolites present in breast

milk. This metabonomic approach, of untargeted profiling of low molecular weight metabolites, in order to characterise breast milk is novel in the field of breast milk research. In this initial report we set out to describe the method development phase of metabolomic characterisation of human breast milk and some preliminary findings on metabolites identified as changing over the time course of lactation.

Methods: The study had Research Ethics approval (10/H0713/5) and informed consent was obtained from the mothers. Human milk samples (5ml) were obtained from 64 mothers of term infants, between 2 to 80 days postpartum. Samples were stored at -80°C until analysis. Milk samples were Folch extracted: 400µl of milk was added to a mixture of 2ml chloroform:methanol (2:1 v/v) and vortexed, following which 600µl of demineralised water was added. The sample was then centrifuged (13000RPM, 10mins). The upper (aqueous phase) and the lower (lipid phase) was pipetted off and speed vacuumed to dryness. The aqueous phase was reconstituted in 600µl D2O phosphate buffer [1]; the lipid phase in 600µl deuterated chloroform. These samples were then transferred to 5mm capillary tubes and

1H NMR spectra were

acquired using a Bruker DRX 600 MHz spectrometer operating at 300 K, using a 1-dimensional NMR pulse sequence - NOESY (nuclear overhauser effect spectroscopy). In total 64 aqueous fractions and 59 lipid fractions were analysed. The resulting

1H NMR spectra were digitalized and imported into Matlab for

phasing and baseline-correction. Spectra were normalized to the total area and data was scaled using unit variance scaling. Multivariate data analysis was carried out using SIMCA-P+ 12.0.1. Data was analysed by Principal Component Analysis (PCA) to establish if there was any clustering of the data in relation to the time point of milk collection. Then data was modelled using PLS-DA and OPLS-DA in order to compare different time points to one another.

Results: In the aqueous phase of the milk, metabolites which were found to cause separation of the groups in relation to time of collection post-partum in the OPLS-DA model were identified. Metabolites identified were lactose, glutamine and glycerophosphocholine which were all increased in intensity compared to the median spectra, with increasing time of lactation. Furthermore, two as yet unidentified metabolites also caused separation in the OPLS-DA model, decreasing in intensity as lactation proceeded. Compared to the aqueous fraction, there was little change in the lipid metabolite profile over time, as analysed to date.

Conclusions: Results to date have agreed with previously reported literature on the changes in breast milk composition, for example, the increase in glycerphosphocholine content with increasing time of lactation [2]. There appears to be less variation in the hydrophobic phase (mainly lipid) in comparison to the aqueous metabolites in the OPLS-DA models. In order to study the changes in lipid composition of milk, more suitable techniques may include GC-MS, which can resolve more detailed information such as the fatty acid composition of the milk.

1H NMR appears to be a promising technique for the investigation of global milk

composition.

References: 1. Beckonert, O., et al. Nat Protoc, 2007. 2(11): p. 2692-703. 2. Ilcol, Y.O., et al. J Nutr Biochem, 2005. 16(8): p. 489-99. Corresponding Author: n.andreas@imperial.ac.uk

Check box to confirm if Presenting Author is a trainee: clinical trainee

basic science trainee

Senior Author supporting presentation on the day of the meeting: Neena Modi

This abstract has not been published elsewhere.

Title: Human neonatal CD4 T cells display distinct functional potentials relative to adults

Authors (Presenting author underlined. If no author is a Society member please provide the name of the member introducing the author to the Society)

DL Gibbons, PF Fleming, ML Michel, R Carr, KL Costeloe and AC Hayday

Institution: Dept Immunobiology, Kings College London, Guys Hospital, London Bridge, London, SE1 9RT, UK, Blizard Institute, Barts and the London School of Medicine and Dentistry, London E1 2AT, UK and Homerton University Hospital NHS Foundation Trust, Homerton Row, London, E9 6SR, UK

Background/ Introduction (to include Hypothesis and Ethics Approval if required)

T cells are divided on the basis of their T cell receptor (TCR) expression. In peripheral blood the main type

of T cells are those that express the TCR. These cells are further divided depending on their expression

of key cytokines: Th1 cells making IFN; Th2 cells making IL4 and IL13; and Th17 cells making IL17. In

preterm and term neonates, Th1 cell functionality appears to be suboptimal which may predispose to

infection. In this study we assessed key cytokine production from preterm T-cells. (Approved by the South

London REC 2 Committee ID 10/H0802/40)

Methods

Weekly blood samples were taken from preterm babies (23 to 30 weeks gestation) on day 14, 21, 28 and

35 of life. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with phorbol 12-

myristate 13-acetate (PMA) and ionomycin in the presence of brefeldin A for 4 hrs. Cells were stained for

surface markers and then analysed for intracellular cytokine production by flow cytometry. Adult PBMCs

were used as controls.

Results (may contain tables and figures)

As expected, we observed the signatory defect in IFN production by Th1 cells in our preterm samples

(n=20). However, we also consistently observed significantly more interleukin-8 (IL8) producing CD4 T cells

in preterm samples compared to adults (p<0.006). These IL8 producing cells were distinct from T cells

making Th1 cytokines (such as IFN) or Th2 cytokines (such as IL4 and IL13) or Th17 cytokines (such as

IL-17) suggesting a novel T cell population.

Conclusions Our data propose an unrecognized subset of CD4 T cells in the neonate programmed for IL8 production

and apparently distinct from conventional Th1/Th2/Th17 CD4 T cells. Like in adults, it was previously

thought that IL8 production in the neonate originated mainly from polymorphonuclear leukocytes and

monocytes where it plays an important role in activation of the innate immune response. Our data, however,

suggest that the neonatal immune system is enriched with a distinct population of T cells capable of

producing large quantities of IL8. Whilst the exact function of these IL8 producing T cells is unclear, their

function may be to activate the innate immune response and protect the neonate while the adaptive

immune response matures.

References (key references should be included) Corresponding Author email address: deena.gibbons@kcl.ac.uk

Check box to confirm if Presenting Author is a trainee: clinical trainee

basic science trainee

Senior Author supporting presentation on the day of the meeting: KL Costeloe

This abstract has not* been published elsewhere

Title: Expansion of Ventricular Indices to Include Extremes of Prematurity

Authors: Shim R, Gnanasekaran R, Boyle M, Tarrant A, Ryan S, McCallion N

Introducing Member of the Neonatal Society: Dr. Cora Doherty

Institution: Neonatal Department, The Rotunda Hospital, Parnell Square, Dublin 1, Ireland.

Background/ Introduction: Intraventricular haemorrhage (IVH) and Post Haemorrhagic Ventricular Dilation (PVHD) are serious

complications in premature infants and are associated with a high risk of disability. These conditions are

monitored by measuring the ventricular size (Levene Index) and by calculating the Ventricular Index (VI) or

the ratio of the distance between the lateral sides of the ventricles and the biparietal diameter. Treatment

decisions are made based on these measurements, which originate from data obtained 30 years ago1.

These centile charts do not include the extremes of gestations that are treated commonly today nor taken in

to account the advances in perinatal and neonatal care over the intervening period. The aim of this study

was to assess the relationship between Levene Index and grouped gestational ages in preterm infants,

including the gestations less than 27 weeks and to establish normal ranges and median values for VI based

on a more current cohort.

Methods:

This was a retrospective study where serial cranial ultrasounds were reviewed of all preterm infants with

gestation under 32 weeks admitted to Rotunda Hospital from January 2009 to December 2011. The data

collected was grouped according to gestational age at the time of scan from 23 weeks to 45 weeks.

Ventricular sizes from cranial ultrasounds were assessed using VI. An abnormal ultrasound was defined as

one with evidence of IVH grade III or IV, PHVD and was excluded from the study.

Results:

From 255 infants, 816 cranial ultrasounds were

reviewed. For Levene Index, the median values

show a general trend of increase as gestation

increases (Fig. 1). The normal ranges for left VI

were 0 – 0.69 and right VI were 0.23 – 0.88.

The median value for both left and right VI was

0.31.

Conclusions: This study shows that there is a slight increase in Levene Index as gestation at the time of scan increases and that the median values for VI did not significantly change with gestational age at scan. These results provide the basis for updated centile charts for current practice. References: Levene MI. Measurement of the growth of the lateral ventricles in preterm infants with real-time ultrasound. Arch Dis Child. 1981;56:900–904. Corresponding Author email address: mijkboyle@yahoo.com Check box to confirm if Presenting Author is a trainee: clinical trainee

Senior Author supporting presentation on the day of the meeting: Prof. Naomi McCallion

This abstract has not been published elsewhere

Title: Therapeutic Hypothermia for Neonatal Encephalopathy in Low- and Middle-income Countries: A Systematic

Review and Meta-analysis Authors: Shreela Pauliah

1, Angie Wade

2, Seetha Shankaran

3, Sudhin Thayyil

1

Institution: 1

Institute for Women’s Health, University College London, UK;2

Paediatric epidemiology and biostatistics,

University College London, UK; 3

Children's Hospital of Michigan and Hutzel Women's Hospital; Wayne State University School of Medicine, USA

Background/ Introduction

Meta-analysis of cooling trials suggests that therapeutic hypothermia alongside optimal neonatal intensive care treatment reduces mortality and long-term neurodisability after neonatal encephalopathy in high-income countries (1). Encephalopathy related to perinatal asphyxia is about 10–20 times more common in low- and middle-income countries (LMIC) than in high-income countries. Annually, approximately 1 million neonatal deaths are caused by perinatal asphyxia in LMIC (2). However, higher incidence of perinatal sepsis, different co-morbidities, sub optimal neonatal intensive care, and lack of effective low technology cooling devices raise concerns about the use of therapeutic hypothermia in LMIC. We performed a systematic review and meta-analysis of safety and efficacy of therapeutic hypothermia after neonatal encephalopathy in LMIC.

Methods

We examined all randomised or quasi-randomised controlled trials comparing selective head or whole body cooling (initiated within six hours of birth, and continued for at least 48 hours), with standard care, in term or near term infants with neonatal encephalopathy, conducted in a LMIC. Primary outcomes were neonatal mortality and moderate or severe disability at ≥ 18 months of age. Secondary outcomes were blood stream infections, coagulopathy or thrombocytopenia requiring blood products, respiratory failure, and hypotension requiring intervention. We used standard Cochrane methodology for literature search (1995 to 2012), data extraction and analysis. Studies without a standard arm were excluded. We used random effects model for meta-analysis, and examined heterogeneity using the I2 test (RevMan version 5.1.4; Copenhagen).

Results

12 studies (4 selective head cooling, 8 whole body cooling) were identified, 5 were excluded due to lack of control arm; thus a total of 546 babies were included in the meta-analysis. A variety of cooling devices, including ice, frozen gel

packs, fans, water bottles, water circulating caps or phase changing materials were used to administer hypothermia. The neonatal mortality was similar in the cooled and standard care infants, risk ratio (RR) 0.74 (95%

confidence interval (CI) 0.44 to 1.25) (Figure). No difference in blood stream positive infections was seen RR 0.98 (95% CI 0.26 to 3.6). Data were inadequate to examine the long-term neurodevelopmental outcome or other secondary

outcomes. Unlike high-income country cooling trials, most ‘in-trial’ infants had mild or moderate encephalopathy, and were not ventilated. Of note, infants were hypothermic in the standard arm for several hours after birth; conversely hyperthermia was not seen in standard care infants. Conclusions: Therapeutic hypothermia can be effectively administered using low technology cooling devices outside

the setting of a well-equipped neonatal intensive care unit. Although point estimates suggested a reduction in the neonatal mortality following selective head or whole body cooling in LMIC, this was not statistically significant. No data were available on the long-term neurodevelopmental outcomes. The differences in population co-morbidities of LMIC encephalopathic infants may have influenced the treatment effects of hypothermia. Adequately powered clinical trials are required before cooling can be considered as a therapeutic option in LMIC. One such multi-country LMIC clinical trial – HELIX (Hypothermia for Encephalopathy in Low- Income countries) is expected to start recruitment in the near future. References

1. Neurological outcomes at 18 months of age after moderate hypothermia for perinatal hypoxic ischaemic encephalopathy: synthesis and meta-analysis of trial data. Edward et al: BMJ 2010;340:c363 doi:10.1136/bmj.c363

2. 4 million neonatal deaths: When? Where? Why? Joy E Lawn et al. Lancet 2005; 365: 891–900

Corresponding Author s.thayyil@ucl.ac.uk Author is a trainee Senior Author supporting presentation on the day of the meeting: Dr.S.Thayyil This abstract has not been published elsewhere

Title: Keynote Lecture: Perinatal Gene Therapy

Authors Simon N. Waddington, Reader in Gene Transfer Technology Institution: Institute for Women’s Health Gene Transfer Technology Group University College London

Abstract: The past decade has seen clear clinical benefit of gene therapy in several diseases for which conventional medicine offers no treatment. Several clinical trials using gene therapy for single gene disorders have recruited young patients since older subjects may have suffered irrevocable pathological changes or have not survived because of early onset and lethality of the condition. The concept of perinatal gene therapy is an extension of this principle: Diseases in which irreversible changes occur very early in life may be prevented by gene supplementation, or repair, in the fetus, in the associated maternal tissues or in the neonate. Several recent preclinical studies in small and large animal models lend weight to the feasibility and motivation for very early gene therapy intervention

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HI HI

(a) (b)

Figure 1. 31

P-MRS and NIRS during HI and recovery; (a) piglet 175; (b) piglet183.

Investigation of brain tissue oxygenation, cytochrome-c-oxidase measured with NIRS and intracellular metabolites measured with MRS during perinatal cerebral hypoxia-ischaemia I. Tachtsidis

a, A. Bainbridge

b, E. Bȁr

a, K. Broad

c, M. Ezzat

c, S. Faulkner

c, D. Price

b, E. Powell

c, D. Thomas

d, E

Cadyb, X. Golay

d, N. Robertson

c

a Dept. of Medical Physics and Bioengineering, University College London, U.K.

bMedical Physics and Bioengineering, UCLH NHS Foundation Trust, U.K.

cInstitute for Women’s Health, University College London, U.K.

dInstitute of Neurology, University College London, U.K.

Background: Hypoxic-ischaemia (HI) neonatal encephalopathy is associated with high mortality and morbidity rates worldwide. Using magnetic resonance spectroscopy (MRS), our piglet model previously defined the biphasic pattern of energy disruption during and after HI [1]. However, the precise relationship between energy failure and cell death are still unclear. To investigate these relationships we have integrated two non-invasive techniques broadband near-infrared spectroscopy (NIRS) and MRS [2]. Broadband NIRS can be used to measure the brain tissue oxygenation and the oxidation state of cytochrome-c-oxidase (Δ[oxCCO]). CCO is the terminal electron acceptor of the mitochondrial electron transfer chain (ETC) which catalyses over 95% of oxygen metabolism. In this study we investigate brain oxygenation, CCO and energy-resource changes during transient HI and recovery using simultaneous broadband NIRS and phosphorus (

31P) MRS in the piglet.

Methods: 22 healthy piglets (aged <24 hr) were anaesthetised and physiologically monitored. Transient cerebral HI (duration 20 minutes) was induced by reducing the inspired oxygenation and reversibly inflating bilateral carotid artery occluders. Using

31P MRS we measured inorganic phosphate (Pi)/epp, phosphocreatine

(PCr)/epp, and nucleotide triphosphate (NTP)/epp (epp=exchangeable phosphate pool=Pi+PCr+3NTP). NIRS measured cerebral concentration changes of oxy-haemoglobin (HbO2) and deoxy-haemoglobin (HHb), and cytochrome-c-oxidase oxidation state changes (Δ[oxCCO]). Results: Simultaneous

31P-MRS

and NIRS results are shown in Figure 1. HI rapidly reduced brain oxygenation as shown by changes in haemoglobin difference (Δ[Hbdiff]=Δ[HbO2]-Δ[HHb])) closely followed by a fall in Δ[oxCCO]. PCr/epp fell, and Pi/epp rose, quickly while NTP/epp was buffered initially and only declined when Δ[oxCCO] was significantly lowered. During recovery, metabolic markers for piglet 175 returned to baseline (Figure 1(a)); but they didnot for piglet 183 (Figure 1(b)) indicating a severe insult. Discussion: During transient HI, CCO becomes reduced due to oxygen depletion; adenosine triphosphate levels are initially preserved by the creatine kinase reaction leading to PCr decline whereas energy utilisation without oxidative phosphorylation leads to increased Pi. During recovery we have observed high association between the NIRS measurement of Δ[oxCCO] and

31P-MRS. Complementary MRS and NIRS enable better understanding of the cerebral metabolic

response to HI and can help evaluate early interventional therapies. References: [1]Lorek A. et al. Pediatr.Res.,36,699-706(1994). [2]Tachtsidis I. et al. Biomedical Optics (BIOMED) 2012 JM3A.27 2012. Corresponding Author: i.tachtsidis@ucl.ac.uk

Title: Comparison of Bayley-2 and Bayley-3 Cognitive and Motor outcome at 18 months in infants with Neonatal Encephalopathy treated with Hypothermia.

Authors Sally Jary,a Andrew Whitelaw,

a Lars Walloe

b and Marianne Thoresen

a,b

Institution:a Neonatal Neuroscience, School of Clinical Science, University of Bristol, Bristol, UK;

bDepartment of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.

Background/ Introduction Clinical trials of neuroprotection for infants with moderate or severe neonatal encephalopathy (NE) have used death or severe disability as trial outcomes with Bayley Scales of Infant Development-2 Index

(1) scores

<70 as part of criteria for severe disability.(2)

Bayley-3(3)

has now replaced Bayley-2 and, in preterm infants, is reported to give higher scores than Bayley-2.

(4, 5) Term infants with NE have a different spectrum of

neurodevelopment(6,7)

as all have brain injury. We hypothesised that fewer infants with NE would have scores < 70 using Bayley-3 than with Bayley-2. If so, this could confuse interpretation of new neonatal trials of NE and historical comparisons. Our aims were to prospectively compare Bayley-2 with corresponding scores in Bayley-3; to investigate the cut-off thresholds for moderate and severe disability in both tests and to derive conversion algorithms to predict Bayley-2 scores from Bayley-3 scores in a well-defined cohort of infants following NE.

Methods Study participants were 61 children born 2007-2010 who fulfilled the entry criteria for therapeutic hypothermia (TH) and for whom complete standardised scores for both Bayley-2 and Bayley-3 were available. Assessment was at median age 18.3 months (16.8-19.7) in a single session by one assessor proficient in the administration of both versions of the test. Bayley-3 scales were administered with any extra items specific to Bayley-2 interspersed as judged appropriate for the individual child to maximise motivation and minimise fatigue. Items with differences in administration were presented in accordance with Bayley-3 instructions but scored according to instructions of each version of the test. Inter-scorer reliability, investigated in 10 infants from video recordings, was 97%. Thresholds for disability were defined as moderate/severe <70; mild 70-84; none > 85. Local ethical approval and parental consent were obtained.

Results Median Bayley-3 Cognitive Composite (CC) score (100 (65-125)) was 9 points higher than median Bayley-2 Mental Developmental Index (MDI) score (91(37-121)). Median Bayey-3 Motor Composite score (100(58-124)) was 18 points higher than median Bayley-2 Psychomotor Developmental Index (PDI) score (82(29-109)). Of the 10 children with Bayley-2 MDI < 70, only 3 had Bayley-3 CC scores <70 and 7/10 had scores <85. Of the11 children with Bayley-2 PDI < 70, only 3 had Bayley-3 MC scores < 70 and 8/11 had scores < 85. The relationship between different scores was explored using correlation analysis and score comparisons with the strongest correlations were used to derive regression equations using linear regression analysis. Predicted Bayley-2 Index scores derived from the equations were found to restore the number of infants classified with moderate/severe (<70) and mild (<85) degrees of developmental delay to similar proportions found using measured Bayley-2 scores. Conclusions Fewer children had scores <70 using Bayley-3 than with Bayley-2. This finding prohibits the direct comparison of Bayley-3 and Bayley-2 scores in infants with NE. We provide linear regression equations for cognitive and motor development to facilitate more direct comparison of Bayley-2 and Bayley-3 outcomes in infants with NE following TH. References

1.The Bayley Scales of Infant Development 2nd

Edition1993 Psych Corp; 2. Azzopardi DV. et al (2009) NEngl JMed 361(14); 3. The Bayley Scales of Infant and Toddler Development 3

rd Edition 2006 Harcourt

Assessment; 4. Vohr BR. et al (2012) J Pediatr 161(2); 5. Moore T.et al (2012) J Pediatr 160(4); 6. Murray DM. et al (2010) DMCN 52(2) 7. van Handel M. et al (2007) Eur J Pediatr 166(7) Corresponding Author email address: sally.jary@bristol.ac.uk

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basic science trainee

Senior Author supporting presentation on the day of the meeting: Professor Marianne Thoresen

This abstract has not been published elsewhere.

Title: Neither Xenon nor Fentanyl Induce Neuroapoptosis in the Newborn Pig Brain,

however Isoflurane does.

Hemmen Sabir MD1, Sarah Bishop MSc

1, Nicki Cohen D Phil

3, Elke Maes

2, Xun Liu PhD

1, John

Dingley MD4, Marianne Thoresen MD, PhD

1,2

Institutions: 1

School of Clinical Sciences, University of Bristol, St Michael’s Hospital, Bristol, United Kingdom;

2

Department of Physiology, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway; 3Neuropathology, School of Clinical Sciences, University of Bristol, Frenchay Hospital, Bristol, United Kingdom;

4Swansea Medical School, Singleton Park, Swansea, United Kingdom

Background: Common general anesthetic agents may harm the developing brain. Some N-

methyl-D-aspartate (NMDA) antagonists may induce neuronal injury and cell death during

brain development. Xenon doubles neuroprotection when combined with therapeutic

hypothermia after hypoxic-ischemic brain injury in newborn animals. This study examines the

neuroapoptotic effect of breathing 50% xenon with high-dose fentanyl for 24h at

normothermia (NT) or hypothermia (HT) or breathing 2% isoflurane only at NT on healthy

newborn pigs.

Methods: There were 31 pigs (<24h old) and 5 treatment groups: (1)24h of 50% inhaled

xenon (Xe50%) with fentanyl sedation at HT (Trec=33.5°C), (2)24h of Xe50% with fentanyl

sedation at NT (Trec=38.5°C), (3)24h of fentanyl sedation at NT, (4)non ventilated juvenile

controls at NT or (5)24h of inhaled 2% Isoflurane at NT. Animals were sacrificed after 24h of

allocated treatment and brains prepared for histology. Pathological cells were assessed in

different brain regions and apoptotic cells were counted after immunostaining (Caspase-3

and TUNEL).

Results: Neuropathological assessment did not show a significant difference in apoptosis for

examined brain regions between treatment groups (1)–(4), but a large increase in apoptosis

when Isoflurane was used. The numbers of apoptotic cells, assessed by Cleaved caspase-3

and TUNEL-staining, were significantly increased in the Isoflurane group for cortex,

hippocampus and white matter. For groups (1)–(4) regression analysis indicated that

hypothermia gave a small, but statistically significant increase in the number of

immunostained cells in cerebral cortex and white matter(p<0.05).

Conclusion: At NT or HT, neither 24h of inhaled 50%Xenon with fentanyl sedation, nor

fentanyl alone, induce neuroapoptosis in the neonatal pig brain. Breathing 2% Isoflurane

does increase neuroapoptosis in neonatal pigs.

Corresponding Author: Marianne.Thoresen@bristol.ac.uk

Presenting Author is a basic science trainee

Senior Author supporting presentation on the day of the meeting: Professor Marianne Thoresen

This abstract has been submitted.

Title: Characterising the early development of human pain processing and behaviour

Authors Fiona Moultrie, Ravi Poorun, Alan Worley, Eleri Adams, Rebeccah Slater.

Institution: University of Oxford Background/ Introduction (to include Hypothesis and Ethics Approval if required)

Newborn infants who require hospitalisation undergo many clinically-essential painful procedures during a critical period of neurodevelopment. To improve the management of pain in neonates we need to advance our understanding of the early neurodevelopmental changes that underlie the beginning of human pain processing and perception. The aim of the study was to record noxious-evoked activity in the brain and spinal cord, together with behavioural responses in order to characterise how these responses are developmentally regulated. We hypothesised that the amplitude of noxious-specific brain activity would be larger in term infants compared with preterm infants, whereas spinal reflex withdrawal activity would be larger and more sustained in preterm infants compared to term infants. Ethics approval was obtained from the National Research Ethics Service (NRES) and the Oxford University Hospitals NHS Trust.

Methods Twenty-two infants were studied on the Newborn Intensive Care and Special Care Unit, John Radcliffe Hospital, Oxford between April and July 2012. Each infant was studied on a single occasion, when they required a clinically-essential heel lance. Electroencephalography (EEG) was used to measure nociceptive-specific brain activity, and surface electromyography (EMG) was used to measure spinal nociceptive reflex withdrawal activity. Brain activity was characterised by measurement of the amplitude of the nociceptive-specific potential. The spinal nociceptive reflex withdrawal was characterised using the root mean square (RMS) of activity post-stimulus. All infants were videoed to record evoked changes in facial expression. Basic summary statistics were analysed in Graphpad Prism.

Results (may contain tables and figures) The 22 participants were clinically stable newborn infants of 29.1–40.1 weeks gestation, (gestation at birth: 24.7–39.6 weeks, postnatal age 2-68 days). One infant was excluded from EEG analysis on account of movement artefact and EMG was not successfully recorded in three infants. Nociceptive-specific brain activity was identified in 15 of 21 infants. The amplitude of the nociceptive-specific potential significantly increased with gestation at study (p=0.0157) and gestation at birth (p=0.0362) but was not dependent on postnatal age (p>0.05). EMG activity was significantly greater following noxious stimulation compared to the background EMG (n=19, p<0.001). Background EMG activity significantly increased with gestation (p= 0.0378) whereas nociceptive-specific spinal reflex withdrawal activity significantly decreased with gestation (p=0.0389). When nociceptive-specific cortical activity was present there was a significant reduction in spinal reflex withdrawal activity (Mann-Witney, p<0.01). In 41% (9/22) of infants a facial response was not observed following noxious stimulation. There was no significant difference in mean EMG activity (p>0.05) or mean EEG activity (p>0.05) between facial responders and non-responders.

Conclusions As infants mature during the neonatal period, they display progressively greater nociceptive-specific brain activity and less spinal reflex withdrawal activity. Lack of change in facial expression or minimal withdrawal of a limb in response to a painful procedure is not indicative of a decrease or absence of nociceptive-specific brain activity. Observed motor behaviour relied upon in clinical practice may therefore not reflect the degree of pain processing occurring at the level of the brain and spinal cord. Corresponding Author email address: f.a.moultrie@gmail.com Check box to confirm if Presenting Author is a trainee: ST1 Clinical Trainee √

Senior Author supporting presentation on the day of the meeting: Dr Eleri Adams This abstract has not been published elsewhere. √

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