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.

Intrauterine inflammation and postnatal intravenous dopamine alter the neurovascular unit in preterm newborn lambs.

BACKGROUND: Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading to long-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine administration in the brain of preterm lambs using cellular and molecular analyses. METHODS: At 119-120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPSDA, n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTLDA, n = 7). Post-mortem brain tissue was collected 3-4 h after birth for immunohistochemistry and RT-qPCR analysis of components of the neurovascular unit. RESULTS: LPS exposure increased vascular leakage in the presence of increased vascular density and remodelling with increased astrocyte "end feet" vessel coverage, together with downregulated mRNA levels of the tight junction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects induced by LPS. CONCLUSION: LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Circ_19038 and lnc-AK016022 synergistically regulate Sirt1 to promote remyelination and alleviate white matter injury in preterm mice.

Maternal inflammation can lead to premature birth and fetal brain damage. CircRNA_19038 and lncRNA-AK016022 have been shown to be significantly reduced in brain tissues of preterm mice, while whether they are involved in the regulation of preterm white matter injury remains to be explored. Pregnant mice were intraperitoneally injected with lipopolysaccharide (LPS) to establish a preterm brain injury model. Healthy mice born at term served as controls. Lentivirus-mediated circ_19038 overexpression vector (LV-circ_19038), LV-lnc-AK016022, LV-Sirt1 and LV-sh-Sirt1 were administered to preterm mice through the ventricles. The expression levels of circ_19038, lnc-AK016022 and Sirt1 in the brain tissues of preterm mice were significantly lower than those of full-term healthy mice, and circ_19038 and lnc-AK016022 were co-localized in the brain tissues. Upregulation of circ_19038 or/and lnc-AK016022 promoted remyelination and alleviated white matter structural damage, neuroinflammation, and long-term cognitive and motor deficits in preterm mice, and the combined effect of circ_19038 and lnc-AK016022 showed better results. Primary mouse neuronal cells were isolated to investigate the regulatory effects of circ_19038 and lnc-AK016022 on Sirt1. Circ_19038 and lnc-AK016022 jointly promoted the expression of Sirt1 by adsorbing miR-1b and miR-328, respectively. Moreover, silencing Sirt1 antagonized the beneficial effects of circ_19038 or/and lnc-AK016022 on brain white matter injury in preterm mice. In conclusion, circ_19038 and lnc-AK016022 synergistically regulated Sirt1 expression to promote remyelination and alleviate white matter injury in preterm mice.

Montelukast reduces grey matter abnormalities and functional deficits in a mouse model of inflammation-induced encephalopathy of prematurity.

Encephalopathy of prematurity (EoP) affects approximately 30% of infants born < 32 weeks gestation and is highly associated with inflammation in the foetus. Here we evaluated the efficacy of montelukast, a cysteinyl leukotriene receptor antagonist widely used to treat asthma in children, to ameliorate peripheral and central inflammation, and subsequent grey matter neuropathology and behaviour deficits in a mouse model of EoP. Male CD-1 mice were treated with intraperitoneal (i.p.) saline or interleukin-1beta (IL-1ß, 40 µg/kg, 5 µL/g body weight) from postnatal day (P)1-5 ± concomitant montelukast (1-30 mg/kg). Saline or montelukast treatment was continued for a further 5 days post-injury. Assessment of systemic and central inflammation and short-term neuropathology was performed from 4 h following treatment through to P10. Behavioural testing, MRI and neuropathological assessments were made on a second cohort of animals from P36 to 54. Montelukast was found to attenuate both peripheral and central inflammation, reducing the expression of pro-inflammatory molecules (IL-1ß, IL-6, TNF) in the brain. Inflammation induced a reduction in parvalbumin-positive interneuron density in the cortex, which was normalised with high-dose montelukast. The lowest effective dose, 3 mg/kg, was able to improve anxiety and spatial learning deficits in this model of inflammatory injury, and alterations in cortical mean diffusivity were not present in animals that received this dose of montelukast. Repurposed montelukast administered early after preterm birth may, therefore, improve grey matter development and outcome in EoP.

The impact of trophic and immunomodulatory factors on oligodendrocyte maturation: Potential treatments for encephalopathy of prematurity.

Encephalopathy of prematurity (EoP) is a major cause of morbidity in preterm neonates, causing neurodevelopmental adversities that can lead to lifelong impairments. Preterm birth-related insults, such as cerebral oxygen fluctuations and perinatal inflammation, are believed to negatively impact brain development, leading to a range of brain abnormalities. Diffuse white matter injury is a major hallmark of EoP and characterized by widespread hypomyelination, the result of disturbances in oligodendrocyte lineage development. At present, there are no treatment options available, despite the enormous burden of EoP on patients, their families, and society. Over the years, research in the field of neonatal brain injury and other white matter pathologies has led to the identification of several promising trophic factors and cytokines that contribute to the survival and maturation of oligodendrocytes, and/or dampening neuroinflammation. In this review, we discuss the current literature on selected factors and their therapeutic potential to combat EoP, covering a wide range of in vitro, preclinical and clinical studies. Furthermore, we offer a future perspective on the translatability of these factors into clinical practice.

In Phantom Validation of Time-Domain Near-Infrared Optical Tomography Pioneer for Imaging Brain Hypoxia and Hemorrhage.

The neonatal brain is a vulnerable organ, and lesions due to hemorrhage and/or ischemia occur frequently in preterm neonates. Even though neuroprotective therapies exist, there is no tool available to detect the ischemic lesions. To address this problem, we have recently designed and built the new time-domain near-infrared optical tomography (TD NIROT) system - Pioneer. Here we present the results of a phantom study of the system performance. We used silicone phantoms to mimic risky situations for brain lesions: hemorrhage and hypoxia. Employing Pioneer, we were able to reconstruct accurately both position and optical properties of these inhomogeneities.

Frontal Occipital and Frontal Temporal Horn Ratios: Comparison and Validation of Head Ultrasound-Derived Indexes With MRI and Ventricular Volumes in Infantile Ventriculomegaly.

OBJECTIVE. The purpose of our study was to assess whether linear ventricular dimensions-specifically, the frontal occipital horn ratio (FOHR) and frontal temporal horn ratio (FTHR) obtained from ultrasound (US)-are reliable measures of ventriculomegaly in infants. Our hypothesis was that these US measures would show good correlation with linear ventricular indexes and ventricular volumes obtained from MRI. MATERIALS AND METHODS. We retrospectively identified 90 infants (age ≤ 6 months corrected gestational age) with ventriculomegaly from 2014 to 2017 who had a total of 100 sets of US and MRI studies performed in a 3-day period. FOHR and FTHR were independently measured on US and MRI by two pediatric radiologists and two pediatric neuroradiologists, respectively. Ventricular and brain volumes were segmented from the MR images, and the ventricle-to-intracranial volume ratio was calculated. MRI served as the reference standard. Intraclass correlation coefficients and Bland-Altman analyses were generated to evaluate interobserver and US-MRI concordance. We assessed correlation of the FOHR and FTHR with the ventricle-to-intracranial volume ratio. RESULTS. Bland-Altman plots of the FOHR and FTHR between US and MRI showed excellent concordance with a bias of 0.05 (95% CI, -0.04 to 0.14) and 0.03 (95% CI, -0.06 to 0.13), respectively. There was good-to-excellent interobserver concordance for FOHR and FTHR on head US or MRI (r = 0.86-0.96). There was good correlation between ventricle-to-intracranial volume ratios and US- and MRI-derived FOHRs and FTHRs (r = 0.79-0.87). CONCLUSION. FOHR and FTHR obtained from US in infants with ventriculomegaly have excellent interobserver concordance, are concordant with MRI-derived linear ratios, and correlate with MRI-derived ventricular volumes. Therefore, US-derived FOHR and FTHR are reliable indexes for clinical follow-up of infantile ventriculomegaly.

Transient Hypoxemia Chronically Disrupts Maturation of Preterm Fetal Ovine Subplate Neuron Arborization and Activity.

Preterm infants are at risk for a broad spectrum of neurobehavioral disabilities associated with diffuse disturbances in cortical growth and development. During brain development, subplate neurons (SPNs) are a largely transient population that serves a critical role to establish functional cortical circuits. By dynamically integrating into developing cortical circuits, they assist in consolidation of intracortical and extracortical circuits. Although SPNs reside in close proximity to cerebral white matter, which is particularly vulnerable to oxidative stress, the susceptibility of SPNs remains controversial. We determined SPN responses to two common insults to the preterm brain: hypoxia-ischemia and hypoxia. We used a preterm fetal sheep model using both sexes that reproduces the spectrum of human cerebral injury and abnormal cortical growth. Unlike oligodendrocyte progenitors, SPNs displayed pronounced resistance to early or delayed cell death from hypoxia or hypoxia-ischemia. We thus explored an alternative hypothesis that these insults alter the maturational trajectory of SPNs. We used DiOlistic labeling to visualize the dendrites of SPNs selectively labeled for complexin-3. SPNs displayed reduced basal dendritic arbor complexity that was accompanied by chronic disturbances in SPN excitability and synaptic activity. SPN dysmaturation was significantly associated with the level of fetal hypoxemia and metabolic stress. Hence, despite the resistance of SPNs to insults that trigger white matter injury, transient hypoxemia disrupted SPN arborization and functional maturation during a critical window in cortical development. Strategies directed at limiting the duration or severity of hypoxemia during brain development may mitigate disturbances in cerebral growth and maturation related to SPN dysmaturation.SIGNIFICANCE STATEMENT The human preterm brain commonly sustains blood flow and oxygenation disturbances that impair cerebral cortex growth and cause life-long cognitive and learning disabilities. We investigated the fate of subplate neurons (SPNs), which are a master regulator of brain development that plays critical roles in establishing cortical connections to other brain regions. We used a preterm fetal sheep model that reproduces key features of brain injury in human preterm survivors. We analyzed the responses of fetal SPNs to transient disturbances in fetal oxygenation. We discovered that SPNs are surprisingly resistant to cell death from low oxygen states but acquire chronic structural and functional changes that suggest new strategies to prevent learning problems in children and adults that survive preterm birth.

Heterogeneous increases of regional cerebral blood flow during preterm brain development: Preliminary assessment with pseudo-continuous arterial spin labeled perfusion MRI.

The human brain develops rapidly during 32-45 postmenstrual weeks (PMW), a critical stage characterized by dramatic increases of metabolic demand. The increasing metabolic demand can be inferred through measurements of regional cerebral blood flow (CBF), which might be coupled to regional metabolism in preterm brains. Arterial spin labeled (ASL) perfusion MRI is one of the few viable approaches for imaging regional CBF of preterm brains, but must be optimized for the extremely slow blood velocity unique in preterm brains. In this study, we explored the spatiotemporal CBF distribution in newborns scanned at the age of 32-45PMW using a pseudo-continuous ASL (pCASL) protocol adapted to slow blood flow in neonates. A total of 89 neonates were recruited. PCASL MRI was acquired from 34 normal newborns and phase contrast (PC) images from 19 newborns. Diffusion tensor images (DTI) were acquired from all 89 neonates for measuring cortical fractional anisotropy (FA), which characterizes cortical microstructure. Reproducible CBF measurements were obtained with the adjusted pCASL sequence. Global CBF measurement based on PC MRI was found to double its value in the 3rd trimester. Regional CBF increases were heterogeneous across the brain with a significantly higher rate of CBF increase in the frontal lobe and a lower rate of CBF increase in the occipital lobe. A significant correlation was found between frontal cortical CBF and cortical FA measurements (p<0.01). Increasing CBF values observed in the frontal lobe corresponded to lower FA values, suggesting that dendritic arborization and synaptic formation might be associated with an elevated local CBF. These results offer a preliminary account of heterogeneous regional CBF increases in a vital early developmental period and may shed the light on underlying metabolic support for cortical microstructural changes during the developmental period of 32-45PMW. Preterm effects and limitations of pCASL techniques in newborns need to be carefully considered for interpretation these results.

Impact of Early Nutritional Intake on Preterm Brain: A Magnetic Resonance Imaging Study.

OBJECTIVES: To investigate the association between early nutritional intake and brain development assessed by magnetic resonance imaging (MRI). STUDY DESIGN: A cohort of neonates born at ≤30 weeks gestational age underwent MRI at term equivalent age. Brain maturation and injury were assessed using the Kidokoro score. Two groups were defined by severity of the scores. The associations between macronutrients intake during the first 2 weeks of life, clinical factors, and imaging scores were analyzed using logistic regression. RESULTS: MRI scores from group 1 patients (n = 27) were normal to mildly abnormal (0-5). Group 2 (n = 15) had more abnormal scores (6-12). The median gestational ages (IQR) were 27.4 (1.9) weeks in group 1 and 27.0 (2.9) weeks in group 2, with birth weights of 900 (318) g (group 1) and 844 (293) g (group 2). In group 2, energy, lipid, and carbohydrate intake were significantly lower than in group 1. Group 2 also showed higher rates of sepsis and clinical risk scores than group 1. After adjustments in bivariate models, higher energy and lipid intake remained significantly associated with improved scores on MRI. This association was stronger for the gray matter component of the score. CONCLUSIONS: Higher energy and lipid intake during the first 2 weeks after birth was associated with a lower incidence of brain lesions and dysmaturation at term equivalent age in preterm neonates.

Controversies in preterm brain injury.

In this review, we highlight critical unresolved questions in the etiology and mechanisms causing preterm brain injury. Involvement of neurons, glia, endogenous factors and exogenous exposures is considered. The structural and functional correlates of interrupted development and injury in the premature brain are under active investigation, with the hope that the cellular and molecular mechanisms underlying developmental abnormalities in the human preterm brain can be understood, prevented or repaired.

Cerebral vascular regulation and brain injury in preterm infants.

Cerebrovascular lesions, mainly germinal matrix hemorrhage and ischemic injury to the periventricular white matter, are major causes of adverse neurodevelopmental outcome in preterm infants. Cerebrovascular lesions and neuromorbidity increase with decreasing gestational age, with the white matter predominantly affected. Developmental immaturity in the cerebral circulation, including ongoing angiogenesis and vasoregulatory immaturity, plays a major role in the severity and pattern of preterm brain injury. Prevention of this injury requires insight into pathogenesis. Cerebral blood flow (CBF) is low in the preterm white matter, which also has blunted vasoreactivity compared with other brain regions. Vasoreactivity in the preterm brain to cerebral perfusion pressure, oxygen, carbon dioxide, and neuronal metabolism is also immature. This could be related to immaturity of both the vasculature and vasoactive signaling. Other pathologies arising from preterm birth and the neonatal intensive care environment itself may contribute to impaired vasoreactivity and ineffective CBF regulation, resulting in the marked variations in cerebral hemodynamics reported both within and between infants depending on their clinical condition. Many gaps exist in our understanding of how neonatal treatment procedures and medications have an impact on cerebral hemodynamics and preterm brain injury. Future research directions for neuroprotective strategies include establishing cotside, real-time clinical reference values for cerebral hemodynamics and vasoregulatory capacity and to demonstrate that these thresholds improve long-term outcomes for the preterm infant. In addition, stimulation of vascular development and repair with growth factor and cell-based therapies also hold promise.

The synergistic effects of mechanical ventilation and intrauterine inflammation on cerebral inflammation in preterm fetal sheep.

Background: Intrauterine inflammation and the requirement for mechanical ventilation independently increase the risk of perinatal brain injury and adverse neurodevelopmental outcomes. We aimed to investigate the effects of mechanical ventilation for 24 h, with and without prior exposure to intrauterine inflammation, on markers of brain inflammation and injury in the preterm sheep brain. Methods: Chronically instrumented fetal sheep at ~115 days of gestation were randomly allocated to receive a single intratracheal dose of 1 mg lipopolysaccharide (LPS) or isovolumetric saline, then further randomly allocated 1 h after to receive mechanical ventilation with room air or no mechanical ventilation (unventilated control + saline [UVC, n = 7]; in utero mechanical ventilation + saline [VENT, n = 8], unventilated control + intratracheal LPS [UVC + LPS, n = 7]; in utero ventilation + intratracheal LPS [VENT + LPS, n = 7]). Serial fetal blood and plasma samples were collected throughout the experimental protocol for assessment of blood biochemistry and plasma interleukin (IL)-6 levels. After 24 h of mechanical ventilation, fetal brains were collected for RT-qPCR and immunohistochemical analyses. Results: LPS exposure increased numbers of microglia and upregulated pro-inflammatory related genes within the cortical gray matter (GM) and subcortical white matter (SCWM) (pLPS < 0.05). Mechanical ventilation alone increased astrocytic cell density in the periventricular white matter (PVWM) (pVENT = 0.03) but had no effect on pro-inflammatory gene expression. The combination of ventilation and LPS increased plasma IL-6 levels (p < 0.02 vs. UVC and VENT groups), and exacerbated expression of pro-inflammatory-related genes (IL1ß, TLR4, PTGS2, CXCL10) and microglial density (p < 0.05 vs. VENT). Conclusion: This study demonstrates that 24 h of mechanical ventilation after exposure to intrauterine inflammation increased markers of systemic and brain inflammation and led to the upregulation of pro-inflammatory genes in the white matter. We conclude that 24 h of mechanical ventilation following intrauterine inflammation may precondition the preterm brain toward being more susceptible to inflammation-induced injury.

Music impacts brain cortical microstructural maturation in very preterm infants: A longitudinal diffusion MR imaging study.

Preterm birth disrupts important neurodevelopmental processes occurring from mid-fetal to term-age. Musicotherapy, by enriching infants' sensory input, might enhance brain maturation during this critical period of activity-dependent plasticity. To study the impact of music on preterm infants' brain structural changes, we recruited 54 very preterm infants randomized to receive or not a daily music intervention, that have undergone a longitudinal multi-shell diffusion MRI acquisition, before the intervention (at 33 weeks' gestational age) and after it (at term-equivalent-age). Using whole-brain fixel-based (FBA) and NODDI analysis (n = 40), we showed a longitudinal increase of fiber cross-section (FC) and fiber density (FD) in all major cerebral white matter fibers. Regarding cortical grey matter, FD decreased while FC and orientation dispersion index (ODI) increased, reflecting intracortical multidirectional complexification and intracortical myelination. The music intervention resulted in a significantly higher longitudinal increase of FC and ODI in cortical paralimbic regions, namely the insulo-orbito-temporopolar complex, precuneus/posterior cingulate gyrus, as well as the auditory association cortex. Our results support a longitudinal early brain macro and microstructural maturation of white and cortical grey matter in preterm infants. The music intervention led to an increased intracortical complexity in regions important for socio-emotional development, known to be impaired in preterm infants.

The neural and cardiovascular effects of exposure of gram-positive bacterial inflammation in preterm fetal sheep.

Perinatal infection or inflammation are associated with adverse neurodevelopmental effects and cardiovascular impairments in preterm infants. Most preclinical studies have examined the effects of gram-negative bacterial inflammation on the developing brain, although gram-positive bacterial infections are a major contributor to adverse outcomes. Killed Su-strain group 3 A streptococcus pyogenes (Picibanil, OK-432) is being used for pleurodesis in fetal hydrothorax/chylothorax. We therefore examined the neural and cardiovascular effects of clinically relevant intra-plural infusions of Picibanil. Chronically instrumented preterm (0.7 gestation) fetal sheep received an intra-pleural injection of low-dose (0.1 mg, n = 8) or high-dose (1 mg, n = 8) Picibanil or saline-vehicle (n = 8). Fetal brains were collected for histology one-week after injection. Picibanil exposure was associated with sustained diffuse white matter inflammation and loss of immature and mature oligodendrocytes and subcortical neurons, and associated loss of EEG power. These neural effects were not dose-dependent. Picibanil was also associated with acute changes in heart rate and attenuation of the maturational increase in mean arterial pressure. Even a single exposure to a low-dose gram-positive bacterial-mediated inflammation during the antenatal period is associated with prolonged changes in vascular and neural function.

Neonatal subarachnoid hemorrhage disrupts multiple aspects of cerebellar development.

Over the past decade, survival rates for extremely low gestational age neonates (ELGANs; <28 weeks gestation) has markedly improved. Unfortunately, a significant proportion of ELGANs will suffer from neurodevelopmental dysfunction. Cerebellar hemorrhagic injury (CHI) has been increasingly recognized in the ELGANs population and may contribute to neurologic dysfunction; however, the underlying mechanisms are poorly understood. To address this gap in knowledge, we developed a novel model of early isolated posterior fossa subarachnoid hemorrhage (SAH) in neonatal mice and investigated both acute and long-term effects. Following SAH on postnatal day 6 (P6), we found significant decreased levels of proliferation with the external granular layer (EGL), thinning of the EGL, decreased Purkinje cell (PC) density, and increased Bergmann glial (BG) fiber crossings at P8. At P42, CHI resulted in decreased PC density, decreased molecular layer interneuron (MLI) density, and increased BG fiber crossings. Results from both Rotarod and inverted screen assays did not demonstrate significant effects on motor strength or learning at P35-38. Treatment with the anti-inflammatory drug Ketoprofen did not significantly alter our findings after CHI, suggesting that treatment of neuro-inflammation does not provide significant neuroprotection post CHI. Further studies are required to fully elucidate the mechanisms through which CHI disrupts cerebellar developmental programming in order to develop therapeutic strategies for neuroprotection in ELGANs.

Cerebral haemodynamic response to somatosensory stimulation in preterm lambs is enhanced following sildenafil and inhaled nitric oxide administration.

Background: Neurovascular coupling (NVC) leads to an increase in local cerebral blood flow and oxygenation in response to increased neural activity and metabolic demand. Impaired or immature NVC reported in the preterm brain, potentially reduces cerebral oxygenation following increased neural activity, predisposing to cerebral tissue hypoxia. Endogenous nitric oxide (NO) is a potent vasodilator and a major mediator of NVC and the cerebral haemodynamic response. NO modulators, such as inhaled nitric oxide (iNO) and sildenafil, induce vasodilation and are used clinically to treat pulmonary hypertension in preterm neonates. However, their impact on NVC in the preterm brain are unknown. We aimed to characterise the cerebral functional haemodynamic response in the preterm brain exposed to NO modulators. We hypothesized that iNO and sildenafil in clinical dosages would increase the baseline cerebral perfusion and the cerebral haemodynamic response to neural activation. Methods: Preterm lambs (126-7 days' gestation) were delivered and mechanically ventilated. The cerebral functional haemodynamic response was measured using near infrared spectroscopy as changes in cerebral oxy- and deoxyhaemoglobin (ΔoxyHb, ΔdeoxyHb), following left median nerve stimulations of 1.8, 4.8, and 7.8 s durations in control preterm lambs (n = 11), and following 4.8 and 7.8 s stimulations in preterm lambs receiving either sildenafil citrate (n = 6, 1.33 mcg/kg/hr) or iNO (n = 8, 20 ppm). Results: Following 1.8, 4.8, and 7.8 s stimulations, ∆oxyHb in the contralateral cortex increased (positive functional response) in 7/11 (64%), 7/11 (64%), and 4/11 (36%) control lambs respectively (p < 0.05). Remaining lambs showed decreased ΔoxyHb (negative functional response). Following 4.8 s stimulations, more lambs receiving sildenafil or iNO (83% and 100% respectively) showed positive functional response compared to the controls (p < 0.05). No significant difference between the three groups was observed at 7.8 s stimulations. Conclusion: In the preterm brain, prolonged somatosensory stimulations increased the incidence of negative functional responses with decreased cerebral oxygenation, suggesting that cerebral oxygen delivery may not match the oxygen demand. Sildenafil and iNO increased the incidence of positive functional responses, potentially enhancing NVC, and cerebral oxygenation.

Activin A rescues preterm brain injury through a novel Noggin/BMP4/Id2 signaling pathway.

Activin A (Act A) has been reported to promote oligodendrocyte progenitor cell (OPC) differentiation in vitro and improve neurological outcomes in adult mice. However, the roles and mechanisms of action of Act A in preterm brain injury are unknown. In the present study, P5 rats were subjected to hypoxia­ischemia to establish a neonatal white matter injury (WMI) model and Act A was injected via the lateral ventricle. Pathological characteristics, OPC differentiation, myelination, and neurological performance were analyzed. Further, the involvement of the Noggin/BMP4/Id2 signaling pathway in the roles of Act A in WMI was explored. Act A attenuated pathological damage, promoted OPC differentiation, enhanced myelin sheath and myelinated axon formation, and improved neurological performance of WMI rats. Moreover, Act A enhanced noggin expression, which, in turn, inhibited the expression of bone morphogenetic protein 4 (BMP4) and inhibitor of DNA binding 2 (Id2). Furthermore, upregulation of Id2 completely abolished the rescue effects of Act A in WMI rats. In conclusion, the present findings suggested that Act A rescues preterm brain injury via targeting a novel Noggin/BMP4/Id2 signaling pathway.

Interleukin-1: an important target for perinatal neuroprotection?

Perinatal inflammation is a significant risk factor for lifelong neurodevelopmental impairments such as cerebral palsy. Extensive clinical and preclinical evidence links the severity and pattern of perinatal inflammation to impaired maturation of white and grey matters and reduced brain growth. Multiple pathways are involved in the pathogenesis of perinatal inflammation. However, studies of human and experimental perinatal encephalopathy have demonstrated a strong causative link between perinatal encephalopathy and excessive production of the pro-inflammatory effector cytokine interleukin-1. In this review, we summarize clinical and preclinical evidence that underpins interleukin-1 as a critical factor in initiating and perpatuating systemic and central nervous system inflammation and subsequent perinatal brain injury. We also highlight the important role of endogenous interleukin-1 receptor antagonist in mitigating interleukin-1-driven neuroinflammation and tissue damage, and summarize outcomes from clinical and mechanistic animal studies that establish the commercially available interleukin-1 receptor antagonist, anakinra, as a safe and effective therapeutic intervention. We reflect on the evidence supporting clinical translation of interleukin-1 receptor antagonist for infants at the greatest risk of perinatal inflammation and impaired neurodevelopment, and suggest a path to advance interleukin-1 receptor antagonist along the translational path for perinatal neuroprotection.