Single-cell atlas reveals meningeal leukocyte heterogeneity in the developing mouse brain.

The meninges are important for brain development and pathology. Using single-cell RNA sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared with their adult counterparts. We also identified novel meningeal microglia-like cell populations that may participate in white matter development. Early after the hypoxic-ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell resolution view of the importance of meningeal leukocytes at the early stage of development in health and disease.

Novel biomarkers of preterm brain injury from blood transcriptome in sheep model of intrauterine asphyxia.

BACKGROUND: Infants born preterm have a higher incidence of neurological deficits. A key step in finding effective treatments is to identify biomarkers that reliably predict outcome. METHODS: Following umbilical cord occlusion (UCO) in pregnant sheep, whole fetal blood RNA was sequenced pre- and post-UCO, brain injury outcome was determined by battery of neuropathology scoring and the transcriptome signature correlated to the degree of brain injury. Additionally, we developed a novel analytical procedure to deduce cell blood composition over time. RESULTS: Sixty-one genes were identified with significant altered expression after UCO. In pre-UCO blood, the level of three mRNAs (Trex2, Znf280b, novel miRNA) and in post-UCO, four mRNAs (Fam184a, Angptl2, novel lincRNA and an unknown protein-coding gene) were associated to brain injury (FDR < 0.01). Several of these mRNAs are related to inflammation and angiogenesis. Pathway analysis highlighted genes playing a role in perinatal death and growth failure. Results also indicate that several leukocyte populations undergo significant changes after UCO. CONCLUSION: We have used a whole transcriptomic approach to uncover novel biomarkers in fetal blood that correlate to neuropathology in the preterm sheep brain. The current data forms a basis for future studies to investigate mechanisms of these mRNAs in the injury progression. IMPACT: Trend analysis of genes following asphyxia reveal a group of genes associated with perinatal death and growth failure. Several pre-asphyxia transcripts were associated to brain injury severity suggesting genomic susceptibility to injury. Several post-asphyxia transcripts were correlated to brain injury severity, thus, serve as potential novel biomarkers of injury outcome. Successfully adaptation of cell profiling algorithms suggests significant changes in blood cell composition following asphyxia.

A follow up on the feasibility after national implementation of magnesium sulfate for neuroprotection prior to preterm birth.

INTRODUCTION: The risk for brain injury manifested as cerebral palsy is higher in very preterm born children than in term. Prenatal administration of magnesium sulfate (MgSO4 ) has been shown to be neuroprotective and reduces the proportion of very preterm born children later diagnosed with cerebral palsy. A Swedish national clinical practice guideline was implemented in March 2020, stipulating the administration of a single intravenous dose of 6 g MgSO4 1-24 h prior to delivery before gestational age 32+0, aiming for 90% treatment coverage. The aim of this study was to evaluate the feasibility of this new clinical practice guideline in the first year of its implementation. MATERIAL AND METHODS: Data on MgSO4 treatment were collected by reviewing the medical charts of women who gave birth to live born children in gestational age 22+0-31+6 during the period of March 1, 2020 to February 28, 2021, at five Swedish university hospitals. Women with pre-eclampsia, eclampsia, or high elevated liver enzymes low platelets (HELLP) were excluded. RESULTS: A total of 388 women were eligible and 79% received treatment with MgSO4 . Of the 21% not receiving treatment, 9% did not receive treatment due to lack of knowledge about the clinical practice guideline, 9% were not possible to treat and 3% had missing data. The proportion treated increased from 72% to 87% from the first to the last 3 months. Of those treated, 81% received the drug within the stipulated timeframe (mean 8.7 h, median 3.4 h). CONCLUSIONS: There was a positive trend over time in the proportion of women receiving MgSO4 treatment, but the a priori target of 90% was not reached during the first year of implementation. Our findings indicate that this target could be reached with additional information to clinicians.

Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia-ischemia.

BACKGROUND: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown. METHODS: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured. RESULTS: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT. CONCLUSIONS: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI. IMPACT: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models.

Induction of labour at 41 weeks of gestation versus expectant management and induction of labour at 42 weeks of gestation: A cost-effectiveness analysis.

OBJECTIVE: To assess the cost-effectiveness of induction of labour (IOL) at 41 weeks of gestation compared with expectant management until 42 weeks of gestation. DESIGN: A cost-effectiveness analysis alongside the Swedish Post-term Induction Study (SWEPIS), a multicentre, randomised controlled superiority trial. SETTING: Fourteen Swedish hospitals during 2016-2018. POPULATION: Women with an uncomplicated singleton pregnancy with a fetus in cephalic position were randomised at 41 weeks of gestation to IOL or to expectant management and induction at 42 weeks of gestation. METHODS: Health benefits were measured in life years and quality-adjusted life years (QALYs) for mother and child. Total cost per birth was calculated, including healthcare costs from randomisation to discharge after delivery, for mother and child. Incremental cost-effectiveness ratios (ICERs) were calculated by dividing the difference in mean cost between the trial arms by the difference in life years and QALYs, respectively. Sampling uncertainty was evaluated using non-parametric bootstrapping. MAIN OUTCOME MEASURES: The cost per gained life year and per gained QALY. RESULTS: The differences in life years and QALYs gained were driven by the difference in perinatal mortality alone. The absolute risk reduction in mortality was 0.004 (from 6/1373 to 0/1373). Based on Swedish life tables, this gives a mean gain in discounted life years and QALYs of 0.14 and 0.12 per birth, respectively. The mean cost per birth was €4108 in the IOL group (n = 1373) and €4037 in the expectant management group (n = 1373), with a mean difference of €71 (95% CI -€232 to €379). The ICER for IOL compared with expectant management was €545 per life year gained and €623 per QALY gained. Confidence intervals were relatively wide and included the possibility that IOL had both lower costs and better health outcomes. CONCLUSIONS: Induction of labour at 41 weeks of gestation results in a better health outcome and no significant difference in costs. IOL is cost-effective compared with expectant management until 42 weeks of gestation using standard threshold values for acceptable cost per life year/QALY. TWEETABLE ABSTRACT: Induction of labour at 41 weeks of gestation is cost-effective compared with expectant management until 42 weeks of gestation.

New possibilities for neuroprotection in neonatal hypoxic-ischemic encephalopathy.

Around 0.75 million babies worldwide suffer from moderate or severe hypoxic-ischemic encephalopathy (HIE) each year resulting in around 400,000 babies with neurodevelopmental impairment. In 2010, neonatal HIE was associated with 2.4% of the total Global Burden of Disease. Therapeutic hypothermia (TH), a treatment that is now standard of care in high-income countries, provides proof of concept that strategies that aim to improve neurodevelopment are not only possible but can also be implemented to clinical practice. While TH is beneficial, neonates with moderate or severe HIE treated with TH still experience devastating complications: 48% (range: 44-53) combined death or moderate/severe disability. There is a concern that TH may not be effective in low- and middle-income countries. Therapies that further improve outcomes are desperately needed, and in high-income countries, they must be tested in conjunction with TH. We have in this review focussed on pharmacological treatment options (e.g. erythropoietin, allopurinol, melatonin, cannabidiol, exendin-4/exenatide). Erythropoietin and allopurinol show promise and are progressing towards the clinic with ongoing definitive phase 3 randomised placebo-controlled trials. However, there remain global challenges for the next decade. Conclusion: There is a need for more optimal animal models, greater industry support/sponsorship, increased use of juvenile toxicology, dose-ranging studies with pharmacokinetic-pharmacodynamic modelling, and well-designed clinical trials to avoid exposure to harmful medications or abandoning putative treatments. What is Known: • Therapeutic hypothermia is beneficial in neonatal hypoxic-ischemic encephalopathy. • Neonates with moderate or severe hypoxic-ischemic encephalopathy treated with therapeutic hypothermia still experience severe sequelae. What is New: • Erythropoietin, allopurinol, melatonin, cannabidiol, and exendin-4/exenatide show promise in conjunction with therapeutic hypothermia. • There is a need for more optimal animal models, greater industry support/sponsorship, increased use of juvenile toxicology, dose-ranging studies with pharmacokinetic-pharmacodynamic modelling, and well-designed clinical trials.

Maternal and fetal serum concentrations of magnesium after administration of a 6-g bolus dose of magnesium sulfate (MgSO4 ) to women with imminent preterm delivery.

INTRODUCTION: Magnesium sulfate is used world-wide to treat pregnant women at imminent risk of preterm delivery in order to protect the brain of the premature infant. Previous research has shown that magnesium sulfate decreases the risk of cerebral palsy by ~30% in infants born preterm. Despite this, the dosage required for optimal neuroprotection remains unknown. We aimed to investigate whether 6 g magnesium sulfate given as a single bolus dose was tolerable for the women and infants and whether the desired target concentration in the mother's blood was reached and non-toxic level in the infant could be ensured. MATERIAL AND METHODS: In total, 49 women who were at risk of delivery prior to 32 weeks of gestation were recruited. They received a bolus dose of 6 g magnesium sulfate intravenously between 1 and 24 h prior to giving birth and were closely monitored during and after infusion. Blood samples from the patients were analyzed at different time-points (20-30 min after start of infusion, 1, 2, 6 and 24 h) post-administration. Blood samples from the umbilical cord were also taken directly after birth to assess the concentration of magnesium in the infant. RESULTS: None of the women who received magnesium sulfate reached serum magnesium concentrations >3.3 mmol/L. In all, 72% of the women showed serum magnesium levels within the therapeutic interval (2.0-3.5 mmol/L) and no adverse events were observed during the infusion. The serum magnesium levels in the mothers declined to pre-bolus-levels within 24 h after delivery. Serum magnesium levels in the umbilical cord samples ranged from 0.87 to 1.4 mmol/L, which means that all but two were within the normal expected range for a newborn premature infant. CONCLUSIONS: A bolus dose of 6 g magnesium sulfate was well tolerated and without any serious side effects in either mother or infant. Most of our women reached the targeted concentration range of serum magnesium levels after infusion was completed. Their infants had magnesium levels within acceptable levels, regardless of gestational week or mother's body mass index.

Neuroprotection offered by mesenchymal stem cells in perinatal brain injury: Role of mitochondria, inflammation, and reactive oxygen species.

Preclinical studies have shown that mesenchymal stem cells have a positive effect in perinatal brain injury models. The mechanisms that cause these neurotherapeutic effects are not entirely intelligible. Mitochondrial damage, inflammation, and reactive oxygen species are considered to be critically involved in the development of injury. Mesenchymal stem cells have immunomodulatory action and exert mitoprotective effects which attenuate production of reactive oxygen species and promote restoration of tissue function and metabolism after perinatal insults. This review summarizes the present state, the underlying causes, challenges and possibilities for effective clinical translation of mesenchymal stem cell therapy.

Serial blood cytokine and chemokine mRNA and microRNA over 48 h are insult specific in a piglet model of inflammation-sensitized hypoxia-ischaemia.

BACKGROUND: Exposure to inflammation exacerbates injury in neonatal encephalopathy (NE). We hypothesized that brain biomarker mRNA, cytokine mRNA and microRNA differentiate inflammation (E. coli LPS), hypoxia (Hypoxia), and inflammation-sensitized hypoxia (LPS+Hypoxia) in an NE piglet model. METHODS: Sixteen piglets were randomized: (i) LPS 2 µg/kg bolus; 1 µg/kg infusion (LPS; n = 5), (ii) Saline with hypoxia (Hypoxia; n = 6), (iii) LPS commencing 4 h pre-hypoxia (LPS+Hypoxia; n = 5). Total RNA was acquired at baseline, 4 h after LPS and 1, 3, 6, 12, 24, 48 h post-insult (animals euthanized at 48 h). Quantitative PCR was performed for cytokines (IL1A, IL6, CXCL8, IL10, TNFA) and brain biomarkers (ENO2, UCHL1, S100B, GFAP, CRP, BDNF, MAPT). MicroRNA was detected using GeneChip (Affymetrix) microarrays. Fold changes from baseline were compared between groups and correlated with cell death (TUNEL) at 48 h. RESULTS: Within 6 h post-insult, we observed increased IL1A, CXCL8, CCL2 and ENO2 mRNA in LPS+Hypoxia and LPS compared to Hypoxia. IL10 mRNA differentiated all groups. Four microRNAs differentiated LPS+Hypoxia and Hypoxia: hsa-miR-23a, 27a, 31-5p, 193-5p. Cell death correlated with TNFA (R = 0.69; p < 0.01) at 1-3 h and ENO2 (R = -0.69; p = 0.01) at 48 h. CONCLUSIONS: mRNA and miRNA differentiated hypoxia from inflammation-sensitized hypoxia within 6 h in a piglet model. This information may inform human studies to enable triage for tailored neuroprotection in NE. IMPACT: Early stratification of infants with neonatal encephalopathy is key to providing tailored neuroprotection. IL1A, CXCL8, IL10, CCL2 and NSE mRNA are promising biomarkers of inflammation-sensitized hypoxia. IL10 mRNA levels differentiated all three pathological states; fold changes from baseline was the highest in LPS+Hypoxia animals, followed by LPS and Hypoxia at 6 h. miR-23, -27, -31-5p and -193-5p were significantly upregulated within 6 h of a hypoxia insult. Functional analysis highlighted the diverse roles of miRNA in cellular processes.

Effect of second-trimester sonographic cervical length on the risk of spontaneous preterm delivery in different risk groups: A prospective observational multicenter study.

INTRODUCTION: The aim of the study is to compare the effect of cervical length measured with transvaginal ultrasound in the second trimester on the risk of spontaneous preterm delivery (PTD) between different risk groups of asymptomatic women with a singleton pregnancy. MATERIAL AND METHODS: This is a pre-planned exploratory analysis of the CERVIX study, a prospective blinded multicenter diagnostic accuracy study. Asymptomatic women with a singleton pregnancy were consecutively recruited at their second-trimester routine ultrasound examination at seven Swedish ultrasound centers. Cervical length was measured with transvaginal ultrasound at 18-20 weeks (Cx1; n = 11 072) and 21-23 weeks (Cx2, optional; n = 6288). The effect of cervical length on the risk of spontaneous PTD and its discriminative ability was compared between women with: (i) previous spontaneous PTD, late miscarriage or cervical conization (high-risk group; n = 1045); (ii) nulliparae without risk factors (n = 5173); (iii) parae without risk factors (n = 4740). Women with previous indicated PTD were excluded (n = 114). Main outcome measures were: effect of cervical length on the risk of spontaneous PTD expressed as odds ratio per 5-mm decrease in cervical length with interaction analysis using logistic regression to test whether the effect differed between groups, area under the receiver operating characteristic curve (AUC), sensitivity, specificity, number needed to screen to detect one spontaneous PTD. RESULTS: The effect of cervical length at Cx2 on the risk of spontaneous PTD <33 weeks was similar in all groups (odds ratios 2.26-2.58, interaction p value 0.91). The discriminative ability at Cx2 was superior to that at Cx1 and was similar in all groups (AUC 0.69-0.76). Cervical length ≤25 mm at Cx2 identified 57% of spontaneous preterm deliveries <33 weeks in the high-risk group with number needed to screen 161. The number needed to screen for groups (ii) and (iii) were 1018 and 843. CONCLUSIONS: The effect of cervical length at 21-23 weeks on the risk of spontaneous PTD <33 weeks is similar in high- and low-risk pregnancies. The differences in number needed to screen should be considered before implementing a screening program.

Efficacy and safety of oral misoprostol vs transvaginal balloon catheter for labor induction: An observational study within the SWEdish Postterm Induction Study (SWEPIS).

INTRODUCTION: Induction of labor is increasing. A common indication for induction of labor is late term and postterm pregnancy at 41 weeks or more. We aimed to evaluate if there are any differences regarding efficacy, safety, and women's childbirth experience between oral misoprostol and transvaginal balloon catheter for cervical ripening in women with a low-risk singleton pregnancy and induction of labor at 41+0 to 42+0 to 1 weeks of gestation. MATERIAL AND METHODS: In this observational study, based on data from the Swedish Postterm Induction Study (SWEPIS), a multicenter randomized controlled trial, a total of 1213 women with a low-risk singleton pregnancy at 41 to 42 weeks of gestation were induced with oral misoprostol (n = 744) or transvaginal balloon catheter (n = 469) at 15 Swedish delivery hospitals. The primary efficacy outcome was vaginal delivery within 24 h and primary safety outcomes were neonatal and maternal composite adverse outcomes. Secondary outcomes included time to vaginal delivery and mode of delivery. Women's childbirth experience was assessed with the Childbirth Experience Questionnaire (CEQ 2.0) and visual analog scale. We present crude and adjusted mean differences and relative risks (RR) with 95% CI. Adjustment was performed for a propensity score based on delivery hospital and baseline characteristics including Bishop score. RESULTS: Vaginal delivery within 24 h was significantly lower in the misoprostol group compared with the balloon catheter group (46.5% [346/744] vs 62.7% [294/469]; adjusted RR 0.76 95% CI 0.640.89]). Primary neonatal and maternal safety outcomes did not differ between groups (neonatal composite 3.5% [36/744] vs 3.2% [15/469]; adjusted RR 0.77 [95% CI 0.31-1.89]; maternal composite 2.3% [17/744] vs 1.9% [9/469]; adjusted RR 1.70 [95% CI 0.58-4.97]). Adjusted mean time to vaginal delivery was increased by 3.8 h (95% CI 1.3-6.2 h) in the misoprostol group. Non-operative vaginal delivery and cesarean delivery rates did not differ. Women's childbirth experience was positive overall and similar in both groups. CONCLUSIONS: Induction of labor with oral misoprostol compared with a transvaginal balloon catheter was associated with a lower probability of vaginal delivery within 24 h and a longer time to vaginal delivery. However, primary safety outcomes, non-operative vaginal delivery, and women's childbirth experience were similar in both groups. Therefore, both methods can be recommended in women with low-risk postdate pregnancies.

Induction of labour at 41 weeks or expectant management until 42 weeks: A systematic review and an individual participant data meta-analysis of randomised trials.

BACKGROUND: The risk of perinatal death and severe neonatal morbidity increases gradually after 41 weeks of pregnancy. Several randomised controlled trials (RCTs) have assessed if induction of labour (IOL) in uncomplicated pregnancies at 41 weeks will improve perinatal outcomes. We performed an individual participant data meta-analysis (IPD-MA) on this subject. METHODS AND FINDINGS: We searched PubMed, Excerpta Medica dataBASE (Embase), The Cochrane Library, Cumulative Index of Nursing and Allied Health Literature (CINAHL), and PsycINFO on February 21, 2020 for RCTs comparing IOL at 41 weeks with expectant management until 42 weeks in women with uncomplicated pregnancies. Individual participant data (IPD) were sought from eligible RCTs. Primary outcome was a composite of severe adverse perinatal outcomes: mortality and severe neonatal morbidity. Additional outcomes included neonatal admission, mode of delivery, perineal lacerations, and postpartum haemorrhage. Prespecified subgroup analyses were conducted for parity (nulliparous/multiparous), maternal age (<35/≥35 years), and body mass index (BMI) (<30/≥30). Aggregate data meta-analysis (MA) was performed to include data from RCTs for which IPD was not available. From 89 full-text articles, we identified three eligible RCTs (n = 5,161), and two contributed with IPD (n = 4,561). Baseline characteristics were similar between the groups regarding age, parity, BMI, and higher level of education. IOL resulted overall in a decrease of severe adverse perinatal outcome (0.4% [10/2,281] versus 1.0% [23/2,280]; relative risk [RR] 0.43 [95% confidence interval [CI] 0.21 to 0.91], p-value 0.027, risk difference [RD] -57/10,000 [95% CI -106/10,000 to -8/10,000], I2 0%). The number needed to treat (NNT) was 175 (95% CI 94 to 1,267). Perinatal deaths occurred in one (<0.1%) versus eight (0.4%) pregnancies (Peto odds ratio [OR] 0.21 [95% CI 0.06 to 0.78], p-value 0.019, RD -31/10,000, [95% CI -56/10,000 to -5/10,000], I2 0%, NNT 326, [95% CI 177 to 2,014]) and admission to a neonatal care unit ≥4 days occurred in 1.1% (24/2,280) versus 1.9% (46/2,273), (RR 0.52 [95% CI 0.32 to 0.85], p-value 0.009, RD -97/10,000 [95% CI -169/10,000 to -26/10,000], I2 0%, NNT 103 [95% CI 59 to 385]). There was no difference in the rate of cesarean delivery (10.5% versus 10.7%; RR 0.98, [95% CI 0.83 to 1.16], p-value 0.81) nor in other important perinatal, delivery, and maternal outcomes. MA on aggregate data showed similar results. Prespecified subgroup analyses for the primary outcome showed a significant difference in the treatment effect (p = 0.01 for interaction) for parity, but not for maternal age or BMI. The risk of severe adverse perinatal outcome was decreased for nulliparous women in the IOL group (0.3% [4/1,219] versus 1.6% [20/1,264]; RR 0.20 [95% CI 0.07 to 0.60], p-value 0.004, RD -127/10,000, [95% CI -204/10,000 to -50/10,000], I2 0%, NNT 79 [95% CI 49 to 201]) but not for multiparous women (0.6% [6/1,219] versus 0.3% [3/1,264]; RR 1.59 [95% CI 0.15 to 17.30], p-value 0.35, RD 27/10,000, [95% CI -29/10,000 to 84/10,000], I2 55%). A limitation of this IPD-MA was the risk of overestimation of the effect on perinatal mortality due to early stopping of the largest included trial for safety reasons after the advice of the Data and Safety Monitoring Board. Furthermore, only two RCTs were eligible for the IPD-MA; thus, the possibility to assess severe adverse neonatal outcomes with few events was limited. CONCLUSIONS: In this study, we found that, overall, IOL at 41 weeks improved perinatal outcome compared with expectant management until 42 weeks without increasing the cesarean delivery rate. This benefit is shown only in nulliparous women, whereas for multiparous women, the incidence of mortality and morbidity was too low to demonstrate any effect. The magnitude of risk reduction of perinatal mortality remains uncertain. Women with pregnancies approaching 41 weeks should be informed on the risk differences according to parity so that they are able to make an informed choice for IOL at 41 weeks or expectant management until 42 weeks. Study Registration: PROSPERO CRD42020163174.

Microbial invasion of the amniotic cavity is associated with impaired cognitive and motor function at school age in preterm children.

BACKGROUND: Chorioamnionitis is an important cause of preterm delivery. Data on neurodevelopmental outcome in exposed infants are inconsistent due to difficulties in diagnosing intrauterine infection/inflammation and lack of detailed long-term follow-up. We investigate cognitive and motor function in preterm infants at early school age and relate the findings to bacteria in amniotic fluid obtained by amniocentesis (microbial invasion of the amniotic cavity (MIAC)) or placenta findings of histological chorioamnionitis (HCA) or fetal inflammatory response syndrome (FIRS). METHOD: Sixty-six infants with gestational age <34 weeks at birth and without major disabilities were assessed using WISC-III and the Bruininks-Oseretsky Test of Motor Proficiency. Results were corrected for gestational age and sex. RESULTS: Children exposed to MIAC had significantly lower scores for full-scale IQ and verbal IQ compared to the non-MIAC group and the difference in full-scale IQ remained after correction for confounding factors. The MIAC group had also significantly lower motor scores after correction. In contrast, motor function was not affected in infants exposed to HCA or FIRS and differences between groups for cognitive scores were lost after corrections. CONCLUSION: Exposure to bacteria in amniotic fluid is associated with lower motor and cognitive scores in school age preterm infants without major disabilities.

Decreased microglial Wnt/ß-catenin signalling drives microglial pro-inflammatory activation in the developing brain.

Microglia of the developing brain have unique functional properties but how their activation states are regulated is poorly understood. Inflammatory activation of microglia in the still-developing brain of preterm-born infants is associated with permanent neurological sequelae in 9 million infants every year. Investigating the regulators of microglial activation in the developing brain across models of neuroinflammation-mediated injury (mouse, zebrafish) and primary human and mouse microglia we found using analysis of genes and proteins that a reduction in Wnt/ß-catenin signalling is necessary and sufficient to drive a microglial phenotype causing hypomyelination. We validated in a cohort of preterm-born infants that genomic variation in the Wnt pathway is associated with the levels of connectivity found in their brains. Using a Wnt agonist delivered by a blood-brain barrier penetrant microglia-specific targeting nanocarrier we prevented in our animal model the pro-inflammatory microglial activation, white matter injury and behavioural deficits. Collectively, these data validate that the Wnt pathway regulates microglial activation, is critical in the evolution of an important form of human brain injury and is a viable therapeutic target.

Second trimester cervical length measurements with transvaginal ultrasound: A prospective observational agreement and reliability study.

INTRODUCTION: Universal screening for preterm delivery by adding transvaginal ultrasound measurement of cervical length to routine second trimester ultrasound has been proposed. The aim is to estimate inter- and intraobserver agreement and reliability of second trimester transvaginal ultrasound measurements of cervical length performed by specially trained midwife sonographers. MATERIAL AND METHODS: This is a prospective reliability and agreement study performed in seven Swedish ultrasound centers. In total, 18 midwife sonographers specially trained to perform ultrasound measurements of cervical length and 286 women in the second trimester were included. In each center, two midwife sonographers measured cervical length a few minutes apart in the same woman, the number of women examined per examiner pair varying between 24 and 30 (LIVE study). Sixteen midwife sonographers measured cervical length twice ≥2 months apart on 93 video clips (CLIPS study). The main outcome measures were mean difference, limits of agreement, intraclass correlation coefficient, intra-individual standard deviation, repeatability, Cohen's kappa and Fleiss kappa. RESULTS: The limits of agreement and intraclass correlation coefficient of the best examiner pair in the LIVE study were -4.06 to 4.72 mm and 0.91, and those of the poorest were -11.11 to 11.39 mm and 0.31. In the CLIPS study, median (range) intra-individual standard deviation was 2.14 mm (1.40-3.46), repeatability 5.93 mm (3.88-9.58), intraclass correlation coefficient 0.84 (0.66-0.94). Median (range) interobserver agreement for cervical length ≤25 mm in the CLIPS study was 94.6% (84.9%-98.9%) and Cohen's kappa 0.56 (0.12-0.92), median (range) intraobserver agreement was 95.2% (87.1%-98.9%) and Cohen's kappa 0.68 (0.27-0.93). CONCLUSIONS: Agreement and reliability of cervical length measurements differed substantially between examiner pairs and examiners. If cervical length measurements are used to guide management there is potential for both over- and under-treatment. Uniform training and rigorous supervision and quality control are advised.

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo.

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro-inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)-expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS-mediated Toll-like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi-1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi-1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi-1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi-1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro-inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation.

γδ T Cells Contribute to Injury in the Developing Brain.

Brain injury in premature infants, especially periventricular leukomalacia, is an important cause of neurologic disabilities. Inflammation contributes to perinatal brain injury development, but the essential mediators that lead to early-life brain injury remain largely unknown. Neonates have reduced capacity for mounting conventional αßT-cell responses. However, γδT cells are already functionally competent during early development and are important in early-life immunity. We investigated the potential contribution of γδT cells to preterm brain injury using postmortem brains from human preterm infants with periventricular leukomalacia and two animal models of preterm brain injury-the hypoxic-ischemic mouse model and a fetal sheep asphyxia model. Large numbers of γδT cells were observed in the brains of mice, sheep, and postmortem preterm infants after injury, and depletion of γδT cells provided protection in the mouse model. The common γδT-cell-associated cytokines interferon-γ and IL-17A were not detectable in the brain. Although there were increased mRNA levels of Il17f and Il22 in the mouse brains after injury, neither IL-17F nor IL-22 cytokines contributed to preterm brain injury. These findings highlight unique features of injury in the developing brain, where, unlike injury in the mature brain, γδT cells function as initiators of injury independently of common γδT-cell-associated cytokines. This finding will help to identify therapeutic targets for preventing or treating preterm infants with brain injury.

Choroid plexus transcriptome and ultrastructure analysis reveals a TLR2-specific chemotaxis signature and cytoskeleton remodeling in leukocyte trafficking.

Perinatal infection and inflammation are major risk factors for injury in the developing brain, however, underlying mechanisms are not fully understood. Leukocyte migration to the cerebrospinal fluid (CSF) and brain is a hallmark of many pathologies of the central nervous system including those in neonates. We previously reported that systemic activation of Toll-like receptor (TLR) 2, a major receptor for gram-positive bacteria, by agonist Pam3CSK4 (P3C) resulted in dramatic neutrophil and monocyte infiltration to the CSF and periventricular brain of neonatal mice, an effect that was absent by the TLR4 agonist, LPS. Here we first report that choroid plexus is a route of TLR2-mediated leukocyte infiltration to the CSF by performing flow cytometry and transmission electron microscopy (TEM) of the choroid plexus. Next, we exploited the striking discrepancy between P3C and LPS effects on cell migration to determine the pathways regulating leukocyte trafficking through the choroid plexus. We performed RNA sequencing on the choroid plexus after administration of P3C and LPS to postnatal day 8 mice. A cluster gene analysis revealed a TLR2-specific signature of chemotaxis represented by 80-fold increased expression of the gene Ccl3 and 1000-fold increased expression of the gene Cxcl2. Ingenuity pathway analysis (IPA) revealed TLR2-specific molecular signaling related to cytoskeleton organization (e.g. actin signaling) as well as inositol phospholipids biosynthesis and degradation. This included upregulation of genes such as Rac2 and Micall2. In support of IPA results, ultrastructural analysis by TEM revealed clefting and perforations in the basement membrane of the choroid plexus epithelial cells in P3C-treated mice. In summary, we show that the choroid plexus is a route of TLR2-mediated transmigration of neutrophils and monocytes to the developing brain, and reveal previously unrecognized mechanisms that includes a specific chemotaxis profile as well as pathways regulating cytoskeleton and basement membrane remodeling.

Neuroprotective exendin-4 enhances hypothermia therapy in a model of hypoxic-ischaemic encephalopathy.

Hypoxic-ischaemic encephalopathy remains a global health burden. Despite medical advances and treatment with therapeutic hypothermia, over 50% of cooled infants are not protected and still develop lifelong neurodisabilities, including cerebral palsy. Furthermore, hypothermia is not used in preterm cases or low resource settings. Alternatives or adjunct therapies are urgently needed. Exendin-4 is a drug used to treat type 2 diabetes mellitus that has also demonstrated neuroprotective properties, and is currently being tested in clinical trials for Alzheimer's and Parkinson's diseases. Therefore, we hypothesized a neuroprotective effect for exendin-4 in neonatal neurodisorders, particularly in the treatment of neonatal hypoxic-ischaemic encephalopathy. Initially, we confirmed that the glucagon like peptide 1 receptor (GLP1R) was expressed in the human neonatal brain and in murine neurons at postnatal Day 7 (human equivalent late preterm) and postnatal Day 10 (term). Using a well characterized mouse model of neonatal hypoxic-ischaemic brain injury, we investigated the potential neuroprotective effect of exendin-4 in both postnatal Day 7 and 10 mice. An optimal exendin-4 treatment dosing regimen was identified, where four high doses (0.5 µg/g) starting at 0 h, then at 12 h, 24 h and 36 h after postnatal Day 7 hypoxic-ischaemic insult resulted in significant brain neuroprotection. Furthermore, neuroprotection was sustained even when treatment using exendin-4 was delayed by 2 h post hypoxic-ischaemic brain injury. This protective effect was observed in various histopathological markers: tissue infarction, cell death, astrogliosis, microglial and endothelial activation. Blood glucose levels were not altered by high dose exendin-4 administration when compared to controls. Exendin-4 administration did not result in adverse organ histopathology (haematoxylin and eosin) or inflammation (CD68). Despite initial reduced weight gain, animals restored weight gain following end of treatment. Overall high dose exendin-4 administration was well tolerated. To mimic the clinical scenario, postnatal Day 10 mice underwent exendin-4 and therapeutic hypothermia treatment, either alone or in combination, and brain tissue loss was assessed after 1 week. Exendin-4 treatment resulted in significant neuroprotection alone, and enhanced the cerebroprotective effect of therapeutic hypothermia. In summary, the safety and tolerance of high dose exendin-4 administrations, combined with its neuroprotective effect alone or in conjunction with clinically relevant hypothermia make the repurposing of exendin-4 for the treatment of neonatal hypoxic-ischaemic encephalopathy particularly promising.

Peripheral myeloid cells contribute to brain injury in male neonatal mice.

BACKGROUND: Neonatal brain injury is increasingly understood to be linked to inflammatory processes that involve specialised CNS and peripheral immune interactions. However, the role of peripheral myeloid cells in neonatal hypoxic-ischemic (HI) brain injury remains to be fully investigated. METHODS: We employed the Lys-EGFP-ki mouse that allows enhanced green fluorescent protein (EGFP)-positive mature myeloid cells of peripheral origin to be easily identified in the CNS. Using both flow cytometry and confocal microscopy, we investigated the accumulation of total EGFP+ myeloid cells and myeloid cell subtypes: inflammatory monocytes, resident monocytes and granulocytes, in the CNS for several weeks following induction of cerebral HI in postnatal day 9 mice. We used antibody treatment to curb brain infiltration of myeloid cells and subsequently evaluated HI-induced brain injury. RESULTS: We demonstrate a temporally biphasic pattern of inflammatory monocyte and granulocyte infiltration, characterised by peak infiltration at 1 day and 7 days after hypoxia-ischemia. This occurs against a backdrop of continuous low-level resident monocyte infiltration. Antibody-mediated depletion of circulating myeloid cells reduced immune cell accumulation in the brain and reduced neuronal loss in male but not female mice. CONCLUSION: This study offers new insight into sex-dependent central-peripheral immune communication following neonatal brain injury and merits renewed interest in the roles of granulocytes and monocytes in lesion development.