BackgroundCaffeine is one of the most frequently administered medicines in neonatology—prescribed for the management of apnoea of prematurity, to aid extubation and increasingly for conditions such as bronchopulmonary dysplasia. Caffeine guidelines for the management of apnoea of prematurity indicate use based on the age of the infant, but this does not account for individual variation in apnoea rate. Consequently, infants may risk caffeine undertreatment or adverse events due to over-exposure. Apnoea in preterm infants is related to nervous system immaturity, hence, as an essential first step to assess whether brain activity may be a useful biomarker for caffeine treatment, we tested the hypothesis that apnoea rate is related to brain activity.MethodsIn this single-centre prospective observational cohort study, we simultaneously recorded brain activity using electroencephalography (EEG) and respiration using impedance pneumography in 74 infants aged 31–36 weeks postmenstrual age (PMA) on 138 separate occasions. The primary outcome was the association between apnoea rate and brain age gap (defined as the difference between the infant's brain age and their PMA; brain age is calculated from brain activity using a deep learning algorithm). In an exploratory sub-study, we compared the apnoea and desaturation rate in the 7 days after infants stopped caffeine treatment, between those infants with immature and mature brain activity.ResultsWe demonstrate that apnoea rate in moderate/late preterm infants is dependent on brain age gap (p:0.024; β [95% CI]:−0.22 [−0.41 to −0.03]). In contrast, apnoea rate was not correlated with PMA (p:0.58; β [95% CI]:−0.04 [−0.16 to 0.09]). In the exploratory sub-study, we find that when caffeine is discontinued, infants with immature brain activity have more frequent apnoeas and desaturations compared with those with more mature brain function.ConclusionsThese findings provide initial evidence to indicate that brain age is a candidate biomarker for personalised caffeine treatment in preterm infants.

Abstract. Although electroencephalography (EEG) is used to assess neonatal pain in research settings, EEG assessments have not been sufficiently characterized for use as end points to assess the efficacy of analgesics in regulatory-endorsed, industry-sponsored trials. We aimed to identify all studies conducted in neonates with EEG recordings during acute somatic nociceptive skin-breaking procedures, and to create a network of authors who will be invited to contribute their individual participant data (IPD) to an IPD meta-analysis to establish the validity, reliability, and clinical interpretability of an EEG-based neonatal pain measure. To identify literature, we searched MEDLINE, Embase, CINAHL, Web of Science, Scopus, Google Scholar, ClinicalTrials.gov, and the WHO ICTRP from database inception to July 2, 2025. Eligible studies were primary empirical studies that included neonates with EEG recordings during acute skin-breaking procedures. We identified 55 studies across 11 countries. Heel lance was the most common painful procedure; others included venipuncture, immunization, and lumbar puncture. The impact of 12 analgesic interventions has been studied to date, mostly nonpharmacological interventions. Individual-electrode EEG is more common than EEG caps. We noted relatively high data loss due to EEG data-quality concerns. A wide range of non-EEG pain-relevant measures have been recorded alongside EEG (eg, behavior, vital signs). Coauthorship network analysis highlighted that authors commonly work within discrete authorship hubs, with limited coauthorship across hubs. The predominance of studies was from European and American institutions, which limits generalizability. We conclude that sufficient data are available to undertake an IPD meta-analysis.

There are several major challenges limiting our ability to test analgesic efficacy for treatment of neonatal pain, and progress in analgesic drug studies in neonates has stalled. One significant issue is the reliance of clinical pain assessments on traditional behavioural and vital signs-based measures and the exclusion of novel brain-based biomarkers. In this review protocol, we outline our strategy to assess the reliability, validity, and interpretability of an electroencephalography (EEG)-based response biomarker for assessment of acute somatic nociceptive pain in neonates. To standardise EEG analysis and generate the outcome of interest, we will perform an individual participant data (IPD) meta-analysis using data from neonates aged 34–44-week postmenstrual age that have had EEG recorded during acute somatic nociceptive skin-breaking procedures. Relevant data from both published and grey literature will be identified by searching six databases (MEDLINE, Embase, CINAHL, Web of Science, Scopus, Google Scholar), two clinical trial registry platforms (ClinicalTrials.gov, WHO ICTRP), and by consulting expert opinion. We will assess availability bias, data accuracy, and data quality by cross-referencing provided data with data descriptions in the literature, identifying duplicates and nonsensical values, and extracting quality control metrics. Data will be synthesised via a two-stage IPD meta-analysis using a random effects modelling approach grouped by site. Reliability (inter- and intra-rater) outcomes will be measured as Gwet’s AC1 coefficient. Validity (known-groups and known-stimuli) outcomes will be measured as EEG response magnitude differences between clinically meaningfully different stimuli. Interpretability will be addressed by providing normative values, in both original and standardised units. The purpose of this study is to establish the reliability, validity, and interpretability of a specific EEG-based response biomarker for assessing acute somatic nociceptive pain in neonates. It will provide an overview of available data and how EEG is being used globally to assess acute neonatal pain. If sufficient IPD are made available and the outcome is reliable, valid, and interpretable, this work will support the use of EEG-based outcome measures as primary endpoints in clinical trials assessing analgesic efficacy in neonates. Systematic review registration: The protocol was registered with PROSPERO on 14 July 2023: CRD42023444809.

Medication in hospitalised infants is often prescribed using a one-size-fits-all approach due to lack of clinical biomarkers. Caffeine is one of the most frequently administered medicines in neonatology - prescribed for the management of apnoea of prematurity, to aid extubation and increasingly for conditions such as bronchopulmonary dysplasia. Caffeine guidelines for the management of apnoea of prematurity indicate use based on age of the infant, but this does not account for individual variation in apnoea rate. Consequently, infants may risk caffeine undertreatment or adverse events due to over-exposure. Apnoea in preterm infants is related to brainstem immaturity, hence as an essential first step to assessing whether brain maturity may be a useful biomarker for caffeine treatment, we tested the hypothesis that apnoea rate is related to brain maturity. Using electroencephalography (EEG), we demonstrate that apnoea rate in late preterm infants is dependent on brain maturity, not postmenstrual age. We provide initial evidence that when caffeine is discontinued, infants with immature brain activity have more frequent apnoeas and desaturations compared with those with more mature brain function. These findings indicate that brain maturity (assessed automatically using machine learning) is a candidate biomarker for personalised caffeine treatment in preterm infants. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Wellcome Trust and Royal Society through a Sir Henry Dale Fellowship awarded to CH (grant number: 213486/Z/18/Z). FU is funded by the Commonwealth Scholarship Commission. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The UK National Research Ethics Service gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Due to ethical requirements, the raw data that support the findings of this study are available upon request to the corresponding author (caroline.hartley{at}paediatrics.ox.ac.uk). Data and code availability The processed data (including brain maturity, apnoea rates and predictor variables for each individual) and the codes for the analysis are available at: https://github.com/CoenZandvoort/Brain\_maturity\_respiration

BACKGROUND/AIMS:Retinopathy of prematurity (ROP) screening is vital for early disease detection in very premature infants but can cause physiological instability. This study compares the physiological response to binocular indirect ophthalmoscopy (BIO) with indentation and non-contact ultra-widefield (UWF) retinal imaging in non-ventilated neonates. The impact of the Dandle WRAP, a specialised swaddling aid, on UWF imaging was also assessed. METHODS:This retrospective study included 86 ROP screening events in 66 non-ventilated infants aged 35.3 weeks (range 30.6-44.6). Vital signs were continuously recorded, evaluating immediate (within 15 min) and longer-term (within 12 h) physiological responses. RESULTS:ROP screening significantly increased heart and respiratory rates and decreased oxygen saturation within 15 min of screening. No significant differences in physiological responses were found between BIO and UWF imaging, although there was a trend towards lower maximum heart rate with UWF imaging. The Dandle WRAP did not significantly alter physiological responses but improved the ease and speed of UWF imaging. CONCLUSION:UWF imaging does not increase physiological instability compared to BIO in non-ventilated infants. Specialised swaddling aids may facilitate the imaging procedure. IMPACT:ROP screening can be distressing for premature infants and induce physiological instability during and after the examination. We deployed non-contact ultra-widefield retinal imaging as the default method of ROP screening and show that it induces comparable physiological responses as traditional indirect ophthalmoscopy in non-ventilated babies. Dandle WRAP swaddling facilitated handling and speed of retinal imaging. The study demonstrates that imaging-based ROP screening is safe and efficacious in non-ventilated neonates, and continuous multimodal physiological recordings can provide detailed assessment of the effects of procedures and medications.

Background Infants undergo multiple clinically-required painful procedures during their time in hospital, and there is an increasing desire from both parents and clinical staff to have parents directly involved in their newborn’s pain relief. To avoid biases due to selective analysis and reporting, a clinical trial’s statistical analysis plan (SAP) should be finalised and registered prior to dataset lock and unblinding. Here, we outline the SAP for the Petal trial, which was registered on the ISRCTN registry prior to dataset lock and unblinding. Methods The Petal trial is a multicentre, individually randomised, parallel-group interventional superiority trial. The study involves in-patient neonates born at or after 35+0 weeks gestation with a postnatal age of ≤7 days, in two hospital research sites (John Radcliffe Hospital, Oxford, UK; Royal Devon and Exeter Hospital, Exeter, UK). The primary objective is to investigate the potential efficacy of a non-pharmacological parent-led stroking intervention on reducing the magnitude of neonates’ noxious stimulus-evoked brain activity. The primary outcome is the neonate’s brain activity recorded using electroencephalography (EEG) in response to a heel lance blood sampling procedure. Secondary outcomes include neonatal clinical pain scores and tachycardia, and parental anxiety. The study hypothesis is neonates’ pain responses and parents’ anxiety scores are lower in the intervention group. Randomisation will be via a minimisation algorithm to maintain balance in five prognostic factors. Conclusions Paediatric pain trials have been highlighted by regulatory bodies as an important and challenging topic, with interest increasing in brain imaging outcomes. The Petal trial, to which this SAP relates, is part of a larger effort of establishing a brain-based EEG outcome measure of infant pain for use in clinical trials. This SAP is thus likely to be of interest to those in academia, pharmaceutical companies, and regulatory bodies. Trial registration ClinicalTrials.gov: NCT04901611, 25/05/2021; ISRCTN: ISRCTN14135962, 23/08/2021).

Background:Infants undergo multiple clinically-required painful procedures during their time in hospital, and there is an increasing desire from both parents and clinical staff to have parents directly involved in their newborn's pain relief. To avoid biases due to selective analysis and reporting, a clinical trial's statistical analysis plan (SAP) should be finalised and registered prior to dataset lock and unblinding. Here, we outline the SAP for the Petal trial, which was registered on the ISRCTN registry prior to dataset lock and unblinding. Methods:The Petal trial is a multicentre, individually randomised, parallel-group interventional superiority trial. The study involves in-patient neonates born at or after 35+0 weeks gestation with a postnatal age of ≤7 days, in two hospital research sites (John Radcliffe Hospital, Oxford, UK; Royal Devon and Exeter Hospital, Exeter, UK). The primary objective is to investigate the potential efficacy of a non-pharmacological parent-led stroking intervention on reducing the magnitude of neonates' noxious stimulus-evoked brain activity. The primary outcome is the neonate's brain activity recorded using electroencephalography (EEG) in response to a heel lance blood sampling procedure. Secondary outcomes include neonatal clinical pain scores and tachycardia, and parental anxiety. The study hypothesis is neonates' pain responses and parents' anxiety scores are lower in the intervention group. Randomisation will be via a minimisation algorithm to maintain balance in five prognostic factors. Conclusions:Paediatric pain trials have been highlighted by regulatory bodies as an important and challenging topic, with interest increasing in brain imaging outcomes. The Petal trial, to which this SAP relates, is part of a larger effort of establishing a brain-based EEG outcome measure of infant pain for use in clinical trials. This SAP is thus likely to be of interest to those in academia, pharmaceutical companies, and regulatory bodies. Trial registration:ClinicalTrials.gov: NCT04901611, 25/05/2021; ISRCTN: ISRCTN14135962, 23/08/2021).

Background Touch interventions such as massage and skin -to -skin contact relieve neonatal pain. The Parental touch trial (Petal) aimed to assess whether parental stroking of their baby before a clinically required heel lance, at a speed of approximately 3 cm/s to optimally activate C -tactile nerve fibres, provides effective pain relief. Methods Petal is a multicentre, randomised, parallel -group interventional superiority trial conducted in the John Radcliffe Hospital (Oxford University Hospitals NHS Foundation Trust, Oxford, UK) and the Royal Devon and Exeter Hospital (Royal Devon University Healthcare NHS Foundation Trust, Exeter, UK). Neonates without neurological abnormalities who were born at 35 weeks gestational age or more and required a blood test via a heel lance in the first week of life were randomly assigned (1:1) to receive parental touch for 10 s either before (intervention group) or after (control group) the clinically required heel lance. Randomisation was managed at the Oxford site using a web -based minimisation algorithm with allocation concealment. The primary outcome measure was the magnitude of noxious -evoked brain activity in response to the heel lance measured with electroencephalography (EEG). Secondary outcome measures were Premature Infant Pain Profile -Revised (PIPP-R) score, development of tachycardia, and parental anxiety score. For all outcomes, the per -protocol effect was estimated via complier average causal effect analysis on the full analysis set. The trial is registered on ISRCTN (ISRCTN14135962) and ClinicalTrials.gov (NCT04901611). Findings Between Sept 1, 2021, and Feb 7, 2023, 159 parents were approached to participate in the study, and 112 neonates were included. 56 neonates were randomly assigned to the intervention group of parental stroking before the heel lance and 56 to the control group of parental stroking after the heel lance. The mean of the magnitude of the heel lance -evoked brain activity was 085 arbitrary units (a.u.; SD 070; n=39; a scaled magnitude of 1 a.u. represents the expected mean response to a heel lance in term -aged neonates) in the intervention group and 091 a.u. (SD 076; n=43) in the control group. Therefore, the primary outcome did not differ significantly between groups, with a mean difference of -011 a.u. (lower in intervention group; SD 077; 95% CI -042 to 020; p=038; n=82). No significant difference was observed across secondary outcomes. The PIPP-R difference in means was 110 (higher in intervention group, 95% CI -042 to 261; p=015; n=100); the odds ratio of becoming tachycardic was 208 (95% CI 046 to 946; p=034, n=105) in the intervention group with reference to the control group; and the difference in parental State -Trait Anxiety Inventory -State score was -044 (higher in control group; SD 685; 95% CI -291 to 202; p=072; n=106). One serious adverse event (desaturation) occurred in a neonate randomly assigned to the control group, which was not considered to be related to the study. Interpretation Parental stroking delivered at an optimal speed to activate C -tactile fibres for a duration of 10 s before the painful procedure did not significantly change neonates' magnitude of pain -related brain activity, PIPP-R score, or development of tachycardia. The trial highlighted the challenge of translating an experimental researcher -led tactile intervention into a parent -led approach, and the value of involving parents in their baby's pain management. Funding Wellcome Trust and Bliss. Copyright (c) 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.

Objective. Automated detection of artefact in stimulus-evoked electroencephalographic (EEG) data recorded in neonates will improve the reproducibility and speed of analysis in clinical research compared with manual identification of artefact. Some studies use very short, single-channel epochs of EEG data with little recorded EEG per infant-for example because the clinical vulnerability of the infants limits access for recording. Current artefact-detection methods that perform well on adult data and resting-state and multi-channel data in infants are not suitable for this application. The aim of this study was to create and test an automated method of detecting artefact in single-channel 1500 ms epochs of infant EEG. Approach. A total of 410 epochs of EEG were used, collected from 160 infants of 28-43 weeks postmenstrual age. This dataset-which was balanced to include epochs of background activity and responses to visual, auditory, tactile and noxious stimuli-was presented to seven independent raters, who independently labelled the epochs according to whether or not they were able to visually identify artefacts. The data was split into a training set (340 epochs) and an independent test set (70 epochs). A random forest model was trained to identify epochs as either artefact or not artefact. Main results. This model performs well, achieving a balanced accuracy of 0.81, which is as good as manual review of data. Accuracy was not significantly related to the infant age or type of stimulus. Significance. This method provides an objective tool for automated artefact rejection for short epoch, single-channel EEG in neonates and could increase the utility of EEG in neonates in both the clinical and research setting.

ABSTRACT:Parental involvement in neonatal comfort care is a core component of family-centred care. Yet, parents experience a range of positive and negative feelings when providing pain-relieving interventions for their infants. Parents of infants who participated in the Parental touch trial ( Petal ), a multicentre randomised controlled trial investigating the impact of gentle parental touch on neonatal pain, were asked to complete an anonymous survey. This survey aimed to (1) explore parent-reported motivations in deciding to participate in the Petal trial; (2) understand parent-reported experiences related to trial participation; (3) understand parents' willingness to participate in future studies; and (4) evaluate parent-reported feelings while they were delivering a gentle touch intervention either before or after a clinically necessary blood test. One hundred six parents (1 parent per infant) took part in the survey. Primary motivators for participation were altruistic. Parents most frequently reported that they wanted their child to take part in the research because it has a potential benefit to babies in the future and because they wanted to improve scientific understanding. Parents reported that providing gentle touch to their children during painful procedures was associated with positive emotions, such as feeling "useful" (64%) and "reassured" (53%). Furthermore, nearly all parents (98%) were pleased to have participated in the Petal trial and would consider, or maybe consider, participating in further research studies. These results underscore the importance of structuring trials around parental involvement and providing opportunities for parents to be involved in providing comfort to their infants during necessary painful clinical procedures.

BACKGROUND:Electroencephalography (EEG) can be used in neonates to measure brain activity changes that are evoked by noxious events, such as clinically required immunisations, cannulation and heel lancing for blood tests. EEG provides an alternative approach to infer pain experience in infants compared with more commonly used behavioural and physiological pain assessments. Establishing the generalisability and construct validity of these measures will help corroborate the use of brain-derived outcomes to evaluate the efficacy of new or existing pharmacological and non-pharmacological methods to treat neonatal pain. This study aimed to test whether a measure of noxious-evoked EEG activity called the noxious neurodynamic response function (n-NRF), that was originally derived in a sample of term-aged infants at the Oxford John Radcliffe Hospital, UK, in 2017, can reliably distinguish noxious from non-noxious events in two independent datasets collected at University College London Hospital and at Royal Devon & Exeter Hospital. We aimed to reproduce three published results that use this measure to quantify noxious-evoked changes in brain activity. We used the n-NRF to quantify noxious-evoked brain activity to test (i) whether significantly larger noxious-evoked activity is recorded in response to a clinical heel lance compared to a non-noxious control heel lance procedure; (ii) whether the magnitude of the activity evoked by a noxious heel lance is equivalent in independent cohorts of infants; and (iii) whether the magnitude of the noxious-evoked brain activity increases with postmenstrual age (PMA) in premature infants up to 37 weeks PMA. Positive replication of these studies will build confidence in the use of the n-NRF as a valid and reliable pain-related outcome which could be used to evaluate analgesic efficacy in neonates. The protocol for this study was published following peer review (https://doi.org/10.17605/OSF.IO/ZY9MS). RESULTS:The n-NRF magnitude to a noxious heel lance stimulus was significantly greater than to a non-noxious control heel lance stimulus in both the UCL dataset (n = 60; mean difference .88; 95% confidence interval (CI) .64-1.13; p < .0001) and the Exeter dataset (n = 31; mean difference .31; 95% CI .02-.61; p = .02). The mean magnitude and 90% bootstrap confidence interval of the n-NRF evoked by the heel lance did not meet our pre-defined equivalence bounds of 1.0 ± .2 in either the UCL dataset (n = 72; mean magnitude 1.33; 90% bootstrapped CI 1.18-1.52) or the Exeter dataset (n = 35; mean magnitude .92, 90% bootstrapped CI .74-1.22). The magnitude of the n-NRF to the noxious stimulus was significantly positively correlated with PMA in infants up to 37 weeks PMA (n = 65; one-sided Pearson's R, adjusted for site: .24; 95% CI .06-1.00; p = .03). CONCLUSIONS:We have reproduced in independent datasets the findings that the n-NRF response to a noxious stimulus is significantly greater than to a non-noxious stimulus, and that the noxious-evoked EEG response increases with PMA. The pre-defined equivalence bounds for the mean magnitude of the EEG response were not met, though this might be due to either inter-site differences such as the lack of calibration of devices between sites (a true negative) or underpowering (a false negative). This reproducibility study provides robust evidence that supports the use of the n-NRF as an objective outcome for clinical trials assessing acute nociception in neonates. Use of the n-NRF in this way has the potential to transform the way analgesic efficacy studies are performed.

OBJECTIVE:We investigated whether sensory-evoked cortical potentials could be used to estimate the age of an infant. Such a model could be used to identify infants who deviate from normal neurodevelopment. METHODS:Infants aged between 28- and 40-weeks post-menstrual age (PMA) (166 recording sessions in 96 infants) received trains of visual and tactile stimuli. Neurodynamic response functions for each stimulus were derived using principal component analysis and a machine learning model trained and validated to predict infant age. RESULTS:PMA could be predicted accurately from the magnitude of the evoked responses (training set mean absolute error and 95% confidence intervals: 1.41 [1.14; 1.74] weeks,p = 0.0001; test set mean absolute error: 1.55 [1.21; 1.95] weeks,p = 0.0002). Moreover, we show that their predicted age (their brain age) is correlated with a measure known to relate to maturity of the nervous system and is linked to long-term neurodevelopment. CONCLUSIONS:Sensory-evoked potentials are predictive of age in premature infants and brain age deviations are related to biologically and clinically meaningful individual differences in nervous system maturation. SIGNIFICANCE:This model could be used to detect abnormal development of infants' response to sensory stimuli in their environment and may be predictive of neurodevelopmental outcome.

Recording multimodal responses to sensory stimuli in infants provides an integrative approach to investigate the developing nervous system. Accurate time-locking across modalities is essential to ensure that responses are interpreted correctly, and could also improve clinical care, for example, by facilitating automatic and objective multimodal pain assessment. Here we develop and assess a system to time-lock stimuli (including clinically-required heel lances and experimental visual, auditory and tactile stimuli) to electrophysiological research recordings and data recorded directly from a hospitalised infant's vital signs monitor. The electronic device presented here (that we have called 'the PiNe box') integrates a previously developed system to time-lock stimuli to electrophysiological recordings and can simultaneously time-lock the stimuli to recordings from hospital vital signs monitors with an average precision of 105 ms (standard deviation: 19 ms), which is sufficient for the analysis of changes in vital signs. Our method permits reliable and precise synchronisation of data recordings from equipment with legacy ports such as TTL (transistor-transistor logic) and RS-232, and patient-connected networkable devices, is easy to implement, flexible and inexpensive. Unlike current all-in-one systems, it enables existing hospital equipment to be easily used and could be used for patients of any age. We demonstrate the utility of the system in infants using visual and noxious (clinically-required heel lance) stimuli as representative examples.

Immune function and sensitivity to pain are closely related, but the association between early life inflammation and sensory nervous system development is poorly understood—especially in humans. Here, in term-born infants, we measure brain activity and reflex withdrawal activity (using EEG and EMG) and behavioural and physiological activity (using the PIPP-R score) to assess the impact of suspected early-onset neonatal infection on tactile- and noxious-evoked responses. We present evidence that neonatal inflammation (assessed by measuring C-reactive protein levels) is associated with increased spinal cord excitability and evoked brain activity following both tactile and noxious stimulation. There are early indications that this hyperalgesia could be maintained post-inflammation, supporting pre-clinical reports of early-life immune dysfunction influencing pain sensitivity in adults.

INTRODUCTION:Newborn infants routinely undergo minor painful procedures as part of postnatal care, with infants born sick or premature requiring a greater number of procedures. As pain in early life can have long-term neurodevelopmental consequences and lead to parental anxiety and future avoidance of interventions, effective pain management is essential. Non-pharmacological comfort measures such as breastfeeding, swaddling and sweet solutions are inconsistently implemented and are not always practical or effective in reducing the transmission of noxious input to the brain. Stroking of the skin can activate C-tactile fibres and reduce pain, and therefore could provide a simple and safe parent-led intervention for the management of pain. The trial aim is to determine whether parental touch prior to a painful clinical procedure provides effective pain relief in neonates. METHODS AND ANALYSIS:This is a multicentre randomised controlled trial. A total of 112 neonates born at 35 weeks' gestation or more requiring a blood test in the first week of life will be recruited and randomised to receive parental stroking either preprocedure or postprocedure. We will record brain activity (EEG), cardiac and respiratory dynamics, oxygen saturation and facial expression to provide proxy pain outcome measures. The primary outcome will be the reduction of noxious-evoked brain activity in response to a heel lance. Secondary outcomes will be a reduction in clinical pain scores (Premature Infant Pain Profile-Revised), postprocedural tachycardia and parental anxiety. ETHICS AND DISSEMINATION:The study has been approved by the London-South East Research Ethics Committee (ref: 21/LO/0523). The results will be widely disseminated through peer-reviewed publications, international conferences and via our partner neonatal charities Bliss and Supporting the Sick Newborn And their Parents (SSNAP). If the parental tactile intervention is effective, recommendations will be submitted via the National Health Service clinical guideline adoption process. STUDY STATUS:Commenced September 2021. TRIAL REGISTRATION NUMBER:NCT04901611; 14 135 962.

Despite the high burden of pain experienced by hospitalised neonates, there are few analgesics with proven efficacy. Testing analgesics in neonates is experimentally and ethically challenging and minimising the number of neonates required to demonstrate efficacy is essential. EEG (electroencephalography)-derived measures of noxious-evoked brain activity can be used to assess analgesic efficacy; however, as variability exists in neonate’s responses to painful procedures, large sample sizes are often required. Here, we present an experimental paradigm to account for individual differences in noxious-evoked baseline sensitivity which can be used to improve the design of analgesic trials in neonates. The paradigm is developed and tested across four observational studies using clinical, experimental, and simulated data (92 neonates). We provide evidence of the efficacy of gentle brushing and paracetamol, substantiating the need for randomised controlled trials of these interventions. This work provides an important step towards safe, cost-effective clinical trials of analgesics in neonates.

Pain assessment in preterm infants is challenging as behavioral, autonomic, and neurophysiological measures of pain are reported to be less sensitive and specific than in term infants. Understanding the pattern of preterm infants' noxious-evoked responses is vital to improve pain assessment in this group. This study investigated the discriminability and development of multimodal noxious-evoked responses in infants aged 28-40 weeks postmenstrual age. A classifier was trained to discriminate responses to a noxious heel lance from a nonnoxious control in 47 infants, using measures of facial expression, brain activity, heart rate, and limb withdrawal, and tested in two independent cohorts with a total of 97 infants. The model discriminates responses to the noxious from the nonnoxious procedure with an overall accuracy of 0.76-0.84 and an accuracy of 0.78-0.79 in the 28-31-week group. Noxious-evoked responses have distinct developmental patterns. Heart rate responses increase in magnitude with age, while noxious-evoked brain activity undergoes three distinct developmental stages, including a previously unreported transitory stage consisting of a negative event-related potential between 30 and 33 weeks postmenstrual age. These findings demonstrate that while noxious-evoked responses change across early development, infant responses to noxious and nonnoxious stimuli are discriminable in prematurity.

Background In the absence of verbal communication it is challenging to infer an individual’s sensory and emotional experience. In adults, fMRI has been used to develop multivariate brain activity signatures, which reliably capture elements of human pain experience. We translate whole-brain fMRI signatures that encode pain perception in adults to the newborn infant brain, to advance understanding of functional brain development and pain perception in early life.Methods A cohort of adults (n=10; mean age=28.3 years) and 2 cohorts of healthy infants (Cohort A: n=15; Cohort B: n=22; mean postnatal age=3 days) were stimulated with low intensity nociceptive stimuli (64-512mN) during acquisition of functional MRI data. fMRI pain signatures were applied directly to the adult data and transformed such that they could be applied to the infant brain. In each cohort, we assessed the concordance of the signatures with the brain responses using cosine-similarity scores, and we assessed stimulus intensity encoding of the signature responses using Spearman rank correlation. Brain activity in ‘pro-pain’ and ‘anti-pain’ brain regions were also examined.Findings The Neurologic Pain Signature (NPS), which reflects aspects of nociceptive pain experience, was activated in both the adults and infants, and reliably encoded stimulus intensity. However, the Stimulus Intensity Independent Pain Signature (SIIPS1), which reflects higher-level cognitive modulation of nociceptive input, was only expressed in adults. ‘Pro-pain’ brain regions showed similar activation patterns in adults and infants, whereas, ‘anti-pain’ brain regions exhibited divergent responses.Interpretation Basic intensity encoding of nociceptive information is similar in adults and infants. However, translation of adult brain signatures into infants reveals significant differences in infant cerebral processing of nociceptive information, which may reflect their lack of expectation, motivation and contextualisation. This study expands the use of brain activity pain signatures to non-verbal patients and provides a potential approach to assess analgesic interventions in infancy.Funding This work was funded by Wellcome (Senior Research Fellowship awarded to Prof. Rebeccah Slater) and SSNAP “Support for Sick and Newborn Infants and their Parents” Medical Research Fund (University of Oxford Excellence Fellowship awarded to Dr Eugene Duff).Evidence before this study We searched PubMed for research articles published prior to March 2020 using terms including ‘fMRI’, ‘infant or neonate’, and ‘pain or nociception’ in the title or abstract. Due to the relatively new emergence of this field, and the experimental and analytical challenges involved in studying cerebral processing of pain in the MRI environment in healthy newborn infants, only five fMRI studies have examined infant brain responses to nociceptive input.In a foundational pilot study, Williams et al., applied an experimental noxious stimulus to a single infant, evoking widespread brain activity that included several brain regions involved in pain processing in adults. Goksan et al., subsequently performed an observational cohort study and used regional analyses to compare active brain regions in infants (n=10) and adults (n=10), concluding that the evoked patterns of brain activity were broadly similar in infants and adults. Further follow-up analysis in the infant cohort revealed that the functional connectivity of brain regions involved in descending pain modulation influences the magnitude of pain-related brain activity. Two further studies focused on methodological advances, providing evidence-based recommendations for fMRI acquisition parameters and image processing in order to maximise the quality of infant data, and these methods have been implemented in this study.Added value of this study This study translates validated adult pain fMRI brain signatures to a nonverbal patient population in which the assessment and management of pain presents a significant clinical challenge. Application of fMRI brain signatures to newborn infants expands on previous fMRI studies that provided only qualitative evidence that noxious stimulation commonly activates brain regions in the adult and infant brain. Here we demonstrate that the basic encoding of the sensory discriminative aspects of pain, as represented by the Neurologic Pain Signature (NPS), occurs in both adults and infants, whereas higher-level cognitive modulation of pain, represented by the Stimulus Intensity Independent Pain Signature (SIIPS1) is only present in adults and not observed in infants. The differences in how the immature infant brain processes pain, relative to the mature adult brain, are likely to reflect differences in their expectation, motivation and contextualisation of external events rather than differences in their core nociceptive cerebral processing of pain. This work allows us to use quantitative fMRI observations to make stronger inferences related to pain experience in nonverbal infants.Implications of all the available evidence Behavioural pain scores used in neonatal clinical care offer limited sensitivity and specificity to pain. Neonatal clinical trials that use these scores as outcome measures frequently report a lack of efficacy of common analgesic interventions, resulting in few evidence-based drugs for treating pain. The value of using brain-based neuroimaging markers of pain as a means of providing objective evidence of analgesic efficacy in early proof of concept studies is well recognised in adults, even in the absence of behavioural pain modulation. Similarly, in infants EEG-based measures of noxious-evoked brain activity have been used as outcome measures in clinical trials of analgesics to overcome some of the inherent limitations of using behavioural observations to quantify analgesic efficacy. Considering the successful translation of the Neurologic Pain Signature (NPS) and its sensitivity to analgesic modulation in adults, this novel methodology represents an objective brain-based fMRI approach that could be used to advance the discovery and assessment of analgesic interventions in infancy.### Competing Interest StatementThe authors have declared no competing interest.