Early evolution of glial morphology and inflammatory cytokines following hypoxic-ischemic injury in the newborn piglet brain.

Neuroinflammation is a hallmark of hypoxic-ischemic injury and can be characterized by the activation of glial cells and the expression of inflammatory cytokines and chemokines. Interleukin (IL)-1ß and tumor necrosis factor (TNF)α are among the best-characterized early response cytokines and are often expressed concurrently. Several types of central nervous system cells secrete IL-1ß and TNFα, including microglia, astrocytes, and neurons, and these cytokines convey potent pro-inflammatory actions. Chemokines also play a central role in neuroinflammation by controlling inflammatory cell trafficking. Our aim was to characterise the evolution of early neuroinflammation in the neonatal piglet model of hypoxic-ischemic encephalopathy (HIE). Piglets (< 24 h old) were exposed to HI insult, and recovered to 2, 4, 8, 12 or 24H post-insult. Brain tissue from the frontal cortex and basal ganglia was harvested for assessment of glial cell activation profiles and transcription levels of inflammatory markers in HI piglets with comparison to a control group of newborn piglets. Fluorescence microscopy was used to observe microglia, astrocytes, neurons, degenerating neurons and possibly apoptotic cells, and quantitative polymerase chain reaction was used to measure gene expression of several cytokines and chemokines. HI injury was associated with microglial activation and morphological changes to astrocytes at all time points examined. Gene expression analyses of inflammation-related markers revealed significantly higher expression of pro-inflammatory cytokines tumor necrosis factor-α (TNFα) and interleukin 1 beta (IL-1ß), chemokines cxc-chemokine motif ligand (CXCL)8 and CXCL10, and anti-inflammatory cytokine transforming growth factor (TGF)ß in every HI group, with some region-specific differences noted. No significant difference was observed in the level of C-X-C chemokine receptor (CCR)5 over time. This high degree of neuroinflammation was associated with a reduction in the number of neurons in piglets at 12H and 24H in the frontal cortex, and the putamen at 12H. This reduction of neurons was not associated with increased numbers of degenerating neurons or potentially apoptotic cells. HI injury triggered a robust early neuroinflammatory response associated with a reduction in neurons in cortical and subcortical regions in our piglet model of HIE. This neuroinflammatory response may be targeted using novel therapeutics to reduce neuropathology in our piglet model of neonatal HIE.

Umbilical Cord Blood Cell Clearance Post-Infusion in Immune-Competent Children with Cerebral Palsy.

Umbilical cord blood cells have therapeutic potential for neurological disorders, through a paracrine mechanism of action. A greater understanding of the safety and immunological effects of allogeneic donor cord blood cells in the context of a healthy recipient immune system, such as in cerebral palsy, is needed. This study aimed to determine how quickly donor cord blood cells were cleared from the circulation in children with cerebral palsy who received a single intravenous infusion of 12/12 human leucocyte antigen (HLA)-matched sibling cord blood cells. Twelve participants with cerebral palsy aged 2-12 years received cord blood cell infusions as part of a phase I trial of umbilical blood infusion for cerebral palsy. Digital droplet PCR analysis of DNA copy number variants specific to donor and recipient was used to assess donor DNA clearance at five timepoints post-infusion, a surrogate measure of cell clearance. Donor cells were cleared by 3 months post-infusion in 11/12 participants. When detected, donor DNA was at a fraction of 0.01-0.31% of total DNA with no signs of graft-versus-host disease in any participant. The donor DNA clearance times provided by this study have important implications for understanding the safety of allogeneic cord blood cell infusion for cerebral palsy and translational tissue engineering or regenerative medicine research in other disorders.

Watch me grow integrated (WMG-I): protocol for a cluster randomised controlled trial of a web-based surveillance approach for developmental screening in primary care settings.

INTRODUCTION: The increasing prevalence of developmental disorders in early childhood poses a significant global health burden. Early detection of developmental problems is vital to ensure timely access to early intervention, and universal developmental surveillance is recommended best practice for identifying issues. Despite this, there is currently considerable variation in developmental surveillance and screening between Australian states and territories and low rates of developmental screening uptake by parents. This study aims to evaluate an innovative web-based developmental surveillance programme and a sustainable approach to referral and care pathways, linking primary care general practice (GP) services that fall under federal policy responsibility and state government-funded child health services. METHODS AND ANALYSIS: The proposed study describes a longitudinal cluster randomised controlled trial (c-RCT) comparing a 'Watch Me Grow Integrated' (WMG-I) approach for developmental screening, to Surveillance as Usual (SaU) in GPs. Forty practices will be recruited across New South Wales and Queensland, and randomly allocated into either the (1) WMG-I or (2) SaU group. A cohort of 2000 children will be recruited during their 18-month vaccination visit or opportunistic visit to GP. At the end of the c-RCT, a qualitative study using focus groups/interviews will evaluate parent and practitioner views of the WMG-I programme and inform national and state policy recommendations. ETHICS AND DISSEMINATION: The South Western Sydney Local Health District (2020/ETH01625), UNSW Sydney (2020/ETH01625) and University of Queensland (2021/HE000667) Human Research Ethics Committees independently reviewed and approved this study. Findings will be reported to the funding bodies, study institutes and partners; families and peer-reviewed conferences/publications. TRIAL REGISTRATION NUMBER: ANZCTR12621000680864.

Effect of asthma management with exhaled nitric oxide versus usual care on perinatal outcomes.

INTRODUCTION: Asthma exacerbations in pregnancy are associated with adverse perinatal outcomes. We aimed to determine whether fractional exhaled nitric oxide (F ENO)-based asthma management improves perinatal outcomes compared to usual care. METHODS: The Breathing for Life Trial was a multicentre, parallel-group, randomised controlled trial conducted in six hospital antenatal clinics, which compared asthma management guided by F ENO (adjustment of asthma treatment according to exhaled nitric oxide and symptoms each 6-12 weeks) to usual care (no treatment adjustment as part of the trial). The primary outcome was a composite of adverse perinatal events (preterm birth, small for gestational age (SGA), perinatal mortality or neonatal hospitalisation) assessed using hospital records. Secondary outcomes included maternal asthma exacerbations. Concealed random allocation, stratified by study site and self-reported smoking status was used, with blinded outcome assessment and statistical analysis (intention to treat). RESULTS: Pregnant women with current asthma were recruited; 599 to the control group (608 infants) and 601 to the intervention (615 infants). There were no significant group differences for the primary composite perinatal outcome (152 (25.6%) out of 594 control, 177 (29.4%) out of 603 intervention; OR 1.21, 95% CI 0.94-1.56; p=0.15), preterm birth (OR 1.14, 95% CI 0.78-1.68), SGA (OR 1.06, 95% CI 0.78-1.68), perinatal mortality (OR 3.62, 95% CI 0.80-16.5), neonatal hospitalisation (OR 1.24, 95% CI 0.89-1.72) or maternal asthma exacerbations requiring hospital admission or emergency department presentation (OR 1.19, 95% CI 0.69-2.05). CONCLUSION: F ENO-guided asthma pharmacotherapy delivered by a nurse or midwife in the antenatal clinic setting did not improve perinatal outcomes.

Combined hypothermia and mesenchymal stem cells in animal models of neonatal hypoxic-ischaemic encephalopathy: a systematic review.

BACKGROUND: The objective of this study was to systematically review the literature to determine the effect of combined hypothermia (HTH) and mesenchymal stem cell (MSC) therapy (administered during or immediately before or after HTH) compared with HTH alone on brain injury and neurobehavioural outcomes in animal models of neonatal hypoxic-ischaemic encephalopathy. METHODS: Primary outcomes assessed were neuropathological measures and neurobehavioural measures of brain outcome. Secondary outcomes were brain protein proinflammatory cytokine status. Risk of bias (ROB) was assessed with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) ROB assessment tool. RESULTS: Of 393 studies identified, 3 studies in postnatal day 7 (P7) male Sprague-Dawley rats met the inclusion criteria. Meta-analyses were undertaken for neuropathological measures (apoptotic cells, astrocytes, microglia), neurobehavioral measures (rotarod test and negative geotaxis), and proinflammatory cytokine levels. Two of the three studies scored low or unclear ROB across all measures. Treatment with HTH-MSCs together significantly improved astrocyte optical density by standardised mean difference (SMD) of 0.71 [95% confidence interval (CI) -1.14, -0.28]. No other measures showed significant differences. CONCLUSIONS: There is insufficient preclinical data to confirm the efficacy of combined HTH-MSC therapy over HTH alone. Future studies should utilise a reporting checklist such as in SYRCLE or Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines to improve reporting standards. IMPACT: Very few articles investigating the use of MSCs for the treatment of hypoxic-ischaemic encephalopathy are clinically relevant. Continuing to publish studies in models of hypoxic-ischaemic encephalopathy without the inclusion of HTH therapy does not progress the field towards improved clinical outcomes. This study shows that HTH and MSC therapy improves measures of astrogliosis. More studies are required to establish the efficacy of HTH and MSCs on measures of neuropathology and neurobehavior. The reporting of preclinical data in this space could be improved by using reporting checklists such as the SYRCLE or ARRIVE tools.

Single group multisite safety trial of sibling cord blood cell infusion to children with cerebral palsy: study protocol and rationale.

INTRODUCTION: Cerebral palsy (CP) is the most common physical disability of childhood but has no cure. Stem cells have the potential to improve brain injury and are proposed as a therapy for CP. However, many questions remain unanswered about the most appropriate cell type, timing of infusions, dose required and associated risks. Therefore, human safety and efficacy trials are necessary to progress knowledge in the field. METHODS AND ANALYSIS: This is a single group study with sample size n=12 to investigate safety of single-dose intravenous 12/12 human leucocyte antigen-matched sibling cord blood cell infusion to children with CP aged 1-16 years without immune suppression. The study is similar to a 3+3 design, where the first two groups of participants have severe CP, and the final six participants include children with all motor severities. Children will be monitored for adverse events and the duration that donor cells are detected. Assessments at baseline, 3 and 12 months will investigate safety and preliminary evidence of change in gross motor, fine motor, cognitive and quality of life outcomes. ETHICS AND DISSEMINATION: Full approval was obtained from The Royal Children's Hospital Human Research Ethics Committee, and a clinical trial notification was accepted by Australia's Therapeutic Goods Administration. Participant guardian informed consent will be obtained before any study procedures. The main results of this study will be submitted for publication in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ACTRN12616000403437, NCT03087110.

Neuropathology in intrauterine growth restricted newborn piglets is associated with glial activation and proinflammatory status in the brain.

BACKGROUND: The fetal brain is particularly vulnerable to intrauterine growth restriction (IUGR) conditions evidenced by neuronal and white matter abnormalities and altered neurodevelopment in the IUGR infant. To further our understanding of neurodevelopment in the newborn IUGR brain, clinically relevant models of IUGR are required. This information is critical for the design and implementation of successful therapeutic interventions to reduce aberrant brain development in the IUGR newborn. We utilise the piglet as a model of IUGR as growth restriction occurs spontaneously in the pig as a result of placental insufficiency, making it a highly relevant model of human IUGR. The purpose of this study was to characterise neuropathology and neuroinflammation in the neonatal IUGR piglet brain. METHODS: Newborn IUGR (< 5th centile) and normally grown (NG) piglets were euthanased on postnatal day 1 (P1; < 18 h) or P4. Immunohistochemistry was utilised to examine neuronal, white matter and inflammatory responses, and PCR for cytokine analysis in parietal cortex of IUGR and NG piglets. RESULTS: The IUGR piglet brain displayed less NeuN-positive cells and reduced myelination at both P1 and P4 in the parietal cortex, indicating neuronal and white matter disruption. A concurrent decrease in Ki67-positive proliferative cells and increase in cell death (caspase-3) in the IUGR piglet brain was also apparent on P4. We observed significant increases in the number of both Iba-1-positive microglia and GFAP-positive astrocytes in the white matter in IUGR piglet brain on both P1 and P4 compared with NG piglets. These increases were associated with a change in activation state, as noted by altered glial morphology. This inflammatory state was further evident with increased expression levels of proinflammatory cytokines (interleukin-1ß, tumour necrosis factor-α) and decreased levels of anti-inflammatory cytokines (interleukin-4 and -10) observed in the IUGR piglet brains. CONCLUSIONS: These findings suggest that the piglet model of IUGR displays the characteristic neuropathological outcomes of neuronal and white matter impairment similar to those reported in the IUGR human brain. The activated glial morphology and elevated proinflammatory cytokines is indicative of an inflammatory response that may be associated with neuronal damage and white matter disruption. These findings support the use of the piglet as a pre-clinical model for studying mechanisms of altered neurodevelopment in the IUGR newborn.

Prevention of gestational diabetes: feasibility issues for an exercise intervention in obese pregnant women.

OBJECTIVE: To examine the feasibility of an individualized exercise program to prevent gestational diabetes mellitus (GDM) in obese pregnant women. RESEARCH DESIGN AND METHODS: The study was a pilot randomized controlled trial with obese pregnant women (intervention group, individualized exercise program [n = 25]; control group, usual care [n = 25]). Average weekly energy expenditure (MET hours per week and kilocalories per week) of exercise-specific activity was assessed during pregnancy using the Pregnancy Physical Activity Questionnaire. Fasting glucose and insulin and homeostasis model assessment of insulin resistance (HOMA-IR) were assessed at baseline and 20, 28, and 36 weeks' gestation. RESULTS: Of the women in the intervention group, 16 of 22 (73%) achieved more than 900 kcal/week of exercise-based activity at 28 weeks compared with 8 of 19 women in the control group (42%), P = 0.047. However, insulin resistance (HOMA-IR) did not differ between the groups. CONCLUSION: This intervention was feasible and prompted a modest increase in physical activity. However, we are not confident that this intervention would be sufficient to prevent GDM.

S-adenosyl-L-methionine restores photoreceptor function following acute retinal ischemia.

The survival and function of retinal neurons is dependent on mitochondrial energy generation and its intracellular distribution by creatine kinase. Post ischemic disruption of retinal creatine synthesis, creatine kinase activity, or transport of creatine into neurons may impair retinal function. S-adenosyl-L-methionine (SAMe) is required for creatine synthesis, phosphatidylcholine and glutathione synthesis, and transducin methylation. These reactions are essential for photoreceptor function but may be downregulated after ischemia due to a reduction in SAMe. Our aim was to determine whether administration of SAMe after ischemia could improve retinal function. Unilateral retinal ischemia was induced in adult rats by increasing the intraocular pressure to 110 mm Hg for 60 min. Immediately after the ischemic insult, SAMe was injected into the vitreous (100 microM), followed by oral administration (69 mg/kg/day) for 5 or 10 days. Retinal function (electroretinography), histology, and creatine transporter (CRT-1) expression were analyzed. Photoreceptoral responses (R(mP3), S), rod and cone bipolar cell responses (PII), and oscillatory potentials were reduced by the ischemia/reperfusion insult. Although SAMe treatment ameliorated the ischemia-induced histological damage by day 5, there was no improvement in retinal function and the intensity of CRT-1 labeling in ischemic retinas was markedly reduced. However, 10 days after ischemia, a recovery in CRT-1 immunolabeling was evident and SAMe supplementation significantly restored photoreceptor function and rod PII responses. In conclusion, these data suggest that creatine transport and methylation reactions, such as creatine synthesis, may be compromised by an ischemic insult contributing to retinal dysfunction and injury. Oral SAMe supplementation after retinal ischemia may provide an effective, safe, and accessible neuroprotective strategy.

Use of the Ages and Stages Questionnaire to predict outcome after hypoxic-ischaemic encephalopathy in the neonate.

BACKGROUND: Infants who suffer hypoxic-ischaemic encephalopathy (HIE) at birth are at increased risk of developmental disability. In this at-risk population, reliable, inexpensive and early identification of those children who are likely to require formal developmental assessment and intervention is needed. AIM: To evaluate the ability of the Ages and Stages Questionnaire (ASQ) to detect developmentally delayed children in an Australian population of infants who suffered HIE at birth. METHODS: Fifty-five children who survived HIE were followed until 12-14 months of age. Test characteristics were calculated to examine the ability of the ASQ to appropriately identify developmentally delayed infants against this study's 'gold standard': the Bayley Scales of Infant Development II. RESULTS: Comparing the ASQ with the Bayley Scales of Infant Development II, the questionnaire had the following test characteristics: sensitivity 92%, specificity 95%, positive predictive value 92%, negative predictive value 95% when used to detect severe developmental delay; and sensitivity 67%, specificity 93%, positive predictive value 92%, negative predictive value 68% when used to detect both severe and mild developmental delay. However, the ASQ used at standard cut-offs failed to detect any of the children with mild delay. CONCLUSIONS: The ASQ is extremely effective for the detection of severe developmental delay in children who have suffered HIE at birth. Its capacity to identify those with milder delay is limited. The ability of the test to detect only those with severe developmental delay means that the ASQ is of little value as a screening tool in this population.

Measuring sensorineural disability in preterm children using a public health screening strategy: a randomised controlled trial.

AIM: To assess the efficacy of a preterm-targeted screening programme against the routine Australian National Health Medical Research Council (NHMRC) universal child health screening programme to detect disability in a general practice setting in children born < or =31 weeks gestation at 12-months of age. METHODS: Multi-centred trial involving 202 preterm children randomised to receive the preterm-targeted or NHMRC programme. Primary outcome, correct identification of neurosensory disability by general practitioners assessed against gold standard paediatric assessments. Sensitivity analysis estimated interrater agreement and screening accuracy. Secondary outcomes, post natal depression (PND), parental stress, health service use, screening programme helpfulness and correct identification of levels of disability severity. RESULTS: Of the 195 infants with data on the primary outcome in the preterm-targeted group, their general practitioners correctly identified the disability status of 61/93 (65.6%) children, as compared with 69/102 (67.6%) in the NHMRC group (odds ratios (OR) 0.91 95% confidence interval (CI) 0.50, 1.65). Responses where general practitioners were unsure of a child's disability status were coded as incorrect and not paired for sensitivity analysis. Sensitivity analysis for 180 diagnostic pairs showed fair interrater agreement for both groups (preterm-targeted k = 0.30 vs. NHMRC k = 0.29) with screening test results favouring the preterm-targeted group with greater sensitivity (73% vs. 33%) but lower specificity (70% vs. 92%) resulting in more over referrals (30% vs. 8%); however, these had a significantly lower mean Developmental Quotient (DQ) score compared with non-disabled children. PND scores were higher in preterm-targeted group (OR 1.33 95% CI 0.01, 2.66). CONCLUSION: The preterm-targeted programme used by general practitioners: (i) did not improve overall identification of disability status compared to the NHMRC universal programme (Australian New Zealand Clinical Trails Registry number, ACTRN 12606000472572); however (ii) it did demonstrate greater efficacy as a screening tool in accurately identifying disabled children.

Perioperative predictors of developmental outcome following cardiac surgery in infancy.

At 1 year we assessed the neurodevelopmental outcomes in infants undergoing cardiac surgery, seeking to explore the predictive value of perioperative markers of cerebral injury. We prospectively enrolled 47 neurodevelopmentally normal infants prior to planned cardiac surgery. Postoperative monitoring consisted of 10-channel video synchronised, continuous electroencephalography from 6 to 30 h, Doppler assessment of cerebral blood flow in the anterior cerebral artery at 1, 2, 3 and 5 h, and measurement of serum S-100B at 0 and 24 h. Neurodevelopmental assessments were performed using the second edition of the Bayley Scale of Infant Development. Follow-up at 1 year was available on 35 infants. The mean age of these patients at surgery had been 57 +/- 15 days. We observed clinical seizures in 1 patient, with 3 other patients having electroencephalographic abnormalities. At follow-up of 1 year, neurodevelopmental scores were lower than preoperative scores, with mean mental scores changing from 103 +/- 5 to 94 +/- 13 (p = 0.001), and mean motor scores changing from 99 +/- 8 to 89 +/- 20 (p = 0.004). No association was found between electroencephalographic abnormalities, reduced cerebral blood flow, or elevation of serum S-100B levels and impaired neurodevelopmental outcome at 1 year. Infants with electroencephalographic abnormalities had elevation of the levels of S-100B in the serum (p = 0.02). At 1 year of follow-up, infants undergoing cardiac surgery demonstrated a reduction in the scores achieved using the second edition of the Bayley Scale of Infant Development. They require ongoing assessment of their progress. Electroencephalographic abnormalities, cerebral blood flow, or levels of S-100B in the serum were not useful perioperative markers for predicting a poor neurodevelopmental outcome in the clinical setting.