CSF1R inhibition promotes neuroinflammation and behavioral deficits during graft-versus-host disease in mice.

ABSTRACT: Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognized, in which brain-infiltrating donor major histocompatibility complex (MHC) class II+ bone marrow-derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early after transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients, in which CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signaling after transplant failed to reverse GVHD-induced behavioral changes. Moreover, we observed aberrant behavior in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr-/- grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.

Blood Biomarkers in the Fetally Growth Restricted and Small for Gestational Age Neonate: Associations with Brain Injury.

Fetal growth restriction (FGR) and small for gestational age (SGA) infants have increased risk of mortality and morbidity. Although both FGR and SGA infants have low birthweights for gestational age, a diagnosis of FGR also requires assessments of umbilical artery Doppler, physiological determinants, neonatal features of malnutrition, and in utero growth retardation. Both FGR and SGA are associated with adverse neurodevelopmental outcomes ranging from learning and behavioral difficulties to cerebral palsy. Up to 50% of FGR, newborns are not diagnosed until around the time of birth, yet this diagnosis lacks further indication of the risk of brain injury or adverse neurodevelopmental outcomes. Blood biomarkers may be a promising tool. Defining blood biomarkers indicating an infant's risk of brain injury would provide the opportunity for early detection and therefore earlier support. The aim of this review was to summarize the current literature to assist in guiding the future direction for the early detection of adverse brain outcomes in FGR and SGA neonates. The studies investigated potential diagnostic blood biomarkers from cord and neonatal blood or serum from FGR and SGA human neonates. Results were often conflicting with heterogeneity common in the biomarkers examined, timepoints, gestational age, and definitions of FGR and SGA used. Due to these variations, it was difficult to draw strong conclusions from the results. The search for blood biomarkers of brain injury in FGR and SGA neonates should continue as early detection and intervention is critical to improve outcomes for these neonates.

Effects of fetal growth restriction on the perinatal neurovascular unit and possible treatment targets.

The neurovascular unit (NVU) within the brain is a multicellular unit that synergistically acts to maintain blood-brain barrier function and meet cerebral metabolic demand. Recent studies have indicated disruption to the NVU is associated with neuropathology in the perinatal brain. Infants with fetal growth restriction (FGR) are known to be at increased risk of neurodevelopmental conditions including motor, learning, and behavioural deficits. There are currently no neuroprotective treatments for these conditions. In this review, we analyse large animal studies examining the effects of FGR on the perinatal NVU. These studies show altered vascularity in the FGR brain as well as blood-brain barrier dysfunction due to underlying cellular changes, mediated by neuroinflammation. Neuroinflammation is a key mechanism associated with pathological effects in the FGR brain. Hence, targeting inflammation may be key to preserving the multicellular NVU and providing neuroprotection in FGR. A number of maternal and postnatal therapies with anti-inflammatory components have been investigated in FGR animal models examining targets for amelioration of NVU disruption. Each therapy showed promise by uniquely ameliorating the adverse effects of FGR on multiple aspects of the NVU. The successful implementation of a clinically viable neuroprotective treatment has the potential to improve outcomes for neonates affected by FGR. IMPACT: Disruption to the neurovascular unit is associated with neuropathology in fetal growth restriction. Inflammation is a key mechanism associated with neurovascular unit disruption in the growth-restricted brain. Anti-inflammatory treatments ameliorate adverse effects on the neurovascular unit and may provide neuroprotection.

Brain Outcomes in Runted Piglets: A Translational Model of Fetal Growth Restriction.

Fetal growth restriction (FGR) is associated with long-term neurodevelopmental disabilities including learning and behavioral disorders, autism, and cerebral palsy. Persistent changes in brain structure and function that are associated with developmental disabilities are demonstrated in FGR neonates. However, the mechanisms underlying these changes remain to be determined. There are currently no therapeutic interventions available to protect the FGR newborn brain. With the wide range of long-term neurodevelopmental disorders associated with FGR, the use of an animal model appropriate to investigating mechanisms of injury in the FGR newborn is crucial for the development of effective and targeted therapies for babies. Piglets are ideal animals to explore how perinatal insults affect brain structure and function. FGR occurs spontaneously in the piglet, unlike other animal models that require surgical or chemical intervention, allowing brain outcomes to be studied without the confounding impacts of experimental interventions. The FGR piglet mimics many of the human pathophysiological outcomes associated with FGR including asymmetrical growth restriction with brain sparing. This review will discuss the similarities observed in brain outcomes between the FGR human and FGR piglet from a magnetic resonance imaging in the living and a histological perspective. FGR piglet studies provide the opportunity to determine and track mechanisms of brain injury in a clinically relevant animal model of FGR. Findings from these FGR piglet studies may provide critical information to rapidly translate neuroprotective interventions to clinic to improve outcomes for newborn babies.

Electroencephalographic studies in growth-restricted and small-for-gestational-age neonates.

Foetal growth restriction (FGR) and being born small for gestational age (SGA) are associated with neurodevelopmental delay. Early diagnosis of neurological damage is difficult in FGR and SGA neonates. Electroencephalography (EEG) has the potential as a tool for the assessment of brain development in FGR/SGA neonates. In this review, we analyse the evidence base on the use of EEG for the assessment of neonates with FGR or SGA. We found consistent findings that FGR/SGA is associated with measurable changes in the EEG that present immediately after birth and persist into childhood. Early manifestations of FGR/SGA in the EEG include changes in spectral power, symmetry/synchrony, sleep-wake cycling, and the continuity of EEG amplitude. Later manifestations of FGR/SGA into infancy and early childhood include changes in spectral power, sleep architecture, and EEG amplitude. FGR/SGA infants had poorer neurodevelopmental outcomes than appropriate for gestational age controls. The EEG has the potential to identify FGR/SGA infants and assess the functional correlates of neurological damage. IMPACT: FGR/SGA neonates have significantly different EEG activity compared to AGA neonates. EEG differences persist into childhood and are associated with adverse neurodevelopmental outcomes. EEG has the potential for early identification of brain impairment in FGR/SGA neonates.

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.

Accuracy of Transient Elastography Data Combined With APRI in Detection and Staging of Liver Disease in Pediatric Patients With Cystic Fibrosis.

BACKGROUND & AIMS: Liver disease develops in 15%-72% of patients with cystic fibrosis, and 5%-10% develop cirrhosis or portal hypertension, usually during childhood. Transient elastography (TE) is a noninvasive method to measure liver stiffness. We aimed to validate its accuracy in detection of liver disease and assessment of fibrosis in children with cystic fibrosis. METHODS: We performed a cross-sectional study to evaluate the accuracy of TE in analysis of liver disease in 160 consecutive children who presented with cystic fibrosis (9.0 ± 0.4 years old, 53% male) at a tertiary referral pediatric center in Australia, from 2011 through 2016. Patients were classified as having cystic fibrosis-associated liver disease (CFLD) or cystic fibrosis without liver disease (CFnoLD) based on clinical, biochemical, and imaging features. Fibrosis severity was determined from histologic analysis of dual-pass liver biopsies from children with CFLD, as the reference standard. Data from healthy children without cystic fibrosis (n = 64, controls) were obtained from a separate study. Liver stiffness measurements (LSMs) were made by Fibroscan analysis, using the inter-quartile range/median ≤30% of 10 valid measurements. Children with macronodularity or portal hypertension with heterogeneous changes on ultrasound without available biopsy were assigned to the category of stage F3-F4 fibrosis. RESULTS: LSM was made reliably in 86% of children; accuracy increased with age. LSMs were significantly higher in children with CFLD (10.7 ± 2.4 kPa, n = 33) than with CFnoLD (4.6 ± 0.1 kPa, n = 105) (P < .0001) or controls (4.1 ± 0.1kPa) (P < .0001); LSMs were higher in children with CFnoLD than controls (P < .05). At a cut-off value of 5.55kPa, LSM identified children with CFLD with an area under the receiver operating characteristic (AUROC) curve of 0.82, 70% sensitivity, and 82% specificity (P < .0001). Classification and regression tree models that combined LSM and aspartate aminotransferase to platelet ratio index (APRI) identified children with CFLD with an AUROC curve of 0.89, 87% sensitivity, and 74% specificity (odds ratio, 18.6). LSMs correlated with fibrosis stage in patients with CFLD (r = 0.67, P = .0001). A cut-off value of 8.7kPa differentiated patients with stage F3-F4 fibrosis from patients with stage F1-F2 fibrosis (AUROC, 0.87; 75% sensitivity; 100% specificity, P=.0002). The combination of LSMs and APRI improved the differentiation of patients with F3-F4 fibrosis vs F1-F2 fibrosis (AUROC, 0.92; 83% sensitivity; and 100% specificity (P < .01). CONCLUSIONS: LSMs made by TE accurately detect liver disease in children with cystic fibrosis; diagnostic accuracy increases when LSMs are combined with APRI. LSMs also differentiate between children with cystic fibrosis with mild-moderate fibrosis vs advanced fibrosis.

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.

Therapeutic potential to reduce brain injury in growth restricted newborns.

Brain injury in intrauterine growth restricted (IUGR) infants is a major contributing factor to morbidity and mortality worldwide. Adverse outcomes range from mild learning difficulties, to attention difficulties, neurobehavioral issues, cerebral palsy, epilepsy, and other cognitive and psychiatric disorders. While the use of medication to ameliorate neurological deficits in IUGR neonates has been identified as warranting urgent research for several years, few trials have been reported. This review summarises clinical trials focusing on brain protection in the IUGR newborn as well as therapeutic interventions trialled in animal models of IUGR. Therapeutically targeting mechanisms of brain injury in the IUGR neonate is fundamental to improving long-term neurodevelopmental outcomes. Inflammation is a key mechanism in neonatal brain injury; and therefore an appealing target. Ibuprofen, an anti-inflammatory drug currently used in the preterm neonate, may be a potential therapeutic candidate to treat brain injury in the IUGR neonate. To better understand the potential of ibuprofen and other therapeutic agents to be neuroprotective in the IUGR neonate, long-term follow-up information of neurodevelopmental outcomes must be studied. Where agents such as ibuprofen are shown to be effective, have a good safety profile and are relatively inexpensive, they can be widely adopted and lead to improved outcomes.

Disruption to the 5-HT7 Receptor Following Hypoxia-Ischemia in the Immature Rodent Brain.

It has become increasingly evident the serotonergic (5-hydroxytryptamine, 5-HT) system is an important central neuronal network disrupted following neonatal hypoxic-ischemic (HI) insults. Serotonin acts via a variety of receptor subtypes that are differentially associated with behavioural and cognitive mechanisms. The 5-HT7 receptor is purported to play a key role in epilepsy, anxiety, learning and memory and neuropsychiatric disorders. Furthermore, the 5-HT7 receptor is highly localized in brain regions damaged following neonatal HI insults. Utilising our well-established neonatal HI model in the postnatal day 3 (P3) rat pup we demonstrated a significant decrease in levels of the 5-HT7 protein in the frontal cortex, thalamus and brainstem one week after insult. We also observed a relative decrease in both the cytosolic and membrane fractions of 5-HT7. The 5-HT7 receptor was detected on neurons throughout the cortex and thalamus, and 5-HT cell bodies in the brainstem. However we found no evidence of 5-HT7 co-localisation on microglia or astrocytes. Moreover, minocycline treatment did not significantly prevent the HI-induced reductions in 5-HT7. In conclusion, neonatal HI injury caused significant disruption to 5-HT7 receptors in the forebrain and brainstem. Yet the use of minocycline to inhibit activated microglia, did not prevent the HI-induced changes in 5-HT7 expression.

Transient liver elastography in unsedated control children: Impact of age and intercurrent illness.

AIM: Transient elastography (TE) is a rapid, non-invasive, reproducible assessment of liver fibrosis by liver stiffness measurement (LSM). Uncertainty remains regarding utility in children, unsedated and <6 years of age. The importance of general health at the time of study has not been addressed. We report our experience of TE in unsedated control children, impact of intercurrent illness and using new published reliability criteria. METHODS: From April 2011 to March 2013, 173 studies were performed in unsedated, healthy control children and children with intercurrent illness without detectable liver disease presenting to the Royal Children's Hospital, Brisbane, Australia. LSM reliability was assessed using interquartile range/median (IQR/M ≤ 30%) of 10 valid measurements. RESULTS: A total of 123 (F:M, 52:71) of 173 studies (71.1%) gave reliable results. In children 0-2 years reliability was 36%, and >2 years reliable results were obtained in ~80%. LSM increased with age; 0-2 years (3.5 ± 0.5 kPa), 3-5 years (3.8 ± 0.3 kPa) and 6-11 years (4.1 ± 0.2 kPa) with healthy older children 12-18 years similar to adults (4.5 ± 0.2 kPa). LSM did not vary with gender (female, 4.5 ± 0.2 vs. male, 4.8 ± 0.2 kPa). Children with intercurrent, non-hepatological illnesses had higher LSM (5.2 ± 0.2 kPa (range, 2.8-11.1 kPa)) compared to healthy children ((4.1 ± 0.1 kPa, range, 2.1-6.3 kPa); P = 0.0001). CONCLUSIONS: TE in unsedated children is feasible from infancy but most reliable after 2 years. Intercurrent illness increases LSM; hence, study context is important when interpreting results.

The search for blood biomarkers that indicate risk of adverse neurodevelopmental outcomes in fetal growth restriction.

Fetal growth restriction (FGR) impacts 5%-10% of pregnancies and is associated with increased risk of mortality and morbidity. Although adverse neurodevelopmental outcomes are observed in up to 50% of FGR infants, a diagnosis of FGR does not indicate the level of risk for an individual infant and these infants are not routinely followed up to assess neurodevelopmental outcomes. Identifying FGR infants at increased risk of adverse neurodevelopmental outcomes would greatly assist in providing appropriate support and interventions earlier, resulting in improved outcomes. However, current methods to detect brain injury around the time of birth lack the sensitivity required to detect the more subtle alterations associated with FGR. Blood biomarkers have this potential. This systematic review assessed the current literature on blood biomarkers for identifying FGR infants at increased risk of adverse neurodevelopmental outcomes at >12 months after birth. Four databases were searched from inception to 22 February 2024. Articles were assessed for meeting the inclusion criteria by two reviewers. The quality of the included article was assessed using Quality Assessment of Diagnostic Accuracy Studies-2. A summary of findings is presented as insufficient articles were identified for meta-analysis. Excluding duplicates, 1,368 records were screened with only 9 articles considered for full text review. Only one article met all the inclusion criteria. Quality assessment indicated low risk of bias. Both blood biomarkers investigated in this study, neuron specific enolase and S100B, demonstrated inverse relationships with neurodevelopmental assessments at 2 years. Four studies did not meet all the inclusion criteria yet identified promising findings for metabolites and cytokines which are discussed here. These findings support the need for further research and highlight the potential for blood biomarkers to predict adverse outcomes. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=369242, Identifier CRD42022369242.

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.

Disruption of raphé serotonergic neural projections to the cortex: a potential pathway contributing to remote loss of brainstem neurons following neonatal hypoxic-ischemic brain injury.

Neuronal injury is a key feature of neonatal hypoxic-ischemic (HI) brain injury. However, the mechanisms underpinning neuronal losses, such as in the brainstem, are poorly understood. One possibility is that disrupted neural connections between the cortex and brainstem may compromise the survival of neuronal cell bodies in the brainstem. We investigated whether brainstem raphé serotonergic neurons that project to the cortex are lost after HI. We also tested if neuroinflammation has a role in disrupting brainstem raphé projections. Postnatal day 3 (P3) rats underwent unilateral carotid artery ligation followed by hypoxia (6% oxygen for 30 min). A retrograde tracer, choleratoxin b, was deposited in the motor cortex on P38. On P45 we found that retrogradely labelled neurons in the dorsal raphé dorsal, ventrolateral, interfascicular, caudal and ventral nuclei were lost after P3 HI. All retrogradely labelled neurons in the raphé nuclei were serotonergic. Numbers of retrogradely labelled neurons were also reduced in the ventromedial thalamus and basolateral amygdala. Minocycline treatment (45 mg/kg 2 h post-HI, 22.5 mg/kg daily P4-P9) attenuated losses of retrogradely labelled neurons in the dorsal raphé ventrolateral, interfascicular and ventral raphé nuclei, and the ventromedial thalamus. These results indicate that raphé neurons projecting to the cortex constitute a population of serotonergic neurons that are lost after P3 HI. Furthermore, neuroinflammation has a role in the disruption of raphé and thalamic neural projections. Future studies investigating the cellular mechanisms of axonal degeneration may reveal new targets for interventions to prevent neuronal losses after neonatal HI.

Neurovascular Unit Alterations in the Growth-Restricted Newborn Are Improved Following Ibuprofen Treatment.

The developing brain is particularly vulnerable to foetal growth restriction (FGR) and abnormal neurodevelopment is common in the FGR infant ranging from behavioural and learning disorders to cerebral palsy. No treatment exists to protect the FGR newborn brain. Recent evidence suggests inflammation may play a key role in the mechanism responsible for the progression of brain impairment in the FGR newborn, including disruption to the neurovascular unit (NVU). We explored whether ibuprofen, an anti-inflammatory drug, could reduce NVU disruption and brain impairment in the FGR newborn. Using a preclinical FGR piglet model, ibuprofen was orally administered for 3 days from birth. FGR brains demonstrated a proinflammatory state, with changes to glial morphology (astrocytes and microglia), and blood-brain barrier disruption, assessed by IgG and albumin leakage into the brain parenchyma and a decrease in blood vessel density. Loss of interaction between astrocytic end-feet and blood vessels was evident where plasma protein leakage was present, suggestive of structural deficits to the NVU. T-cell infiltration was also evident in the parenchyma of FGR piglet brains. Ibuprofen treatment reduced the pro-inflammatory response in FGR piglets, reducing the number of activated microglia and enhancing astrocyte interaction with blood vessels. Ibuprofen also attenuated plasma protein leakage, regained astrocytic end-feet interaction around vessels, and decreased T-cell infiltration into the FGR brain. These findings suggest postnatal administration of ibuprofen modulates the inflammatory state, allowing for stronger interaction between vasculature and astrocytic end-feet to restore NVU integrity. Modulation of the NVU improves the FGR brain microenvironment and may be key to neuroprotection.

Combination of human endothelial colony-forming cells and mesenchymal stromal cells exert neuroprotective effects in the growth-restricted newborn.

The foetal brain is particularly vulnerable to the detrimental effects of foetal growth restriction (FGR) with subsequent abnormal neurodevelopment being common. There are no current treatments to protect the FGR newborn from lifelong neurological disorders. This study examines whether pure foetal mesenchymal stromal cells (MSC) and endothelial colony-forming cells (ECFC) from the human term placenta are neuroprotective through modulating neuroinflammation and supporting the brain vasculature. We determined that one dose of combined MSC-ECFCs (cECFC; 106 ECFC 106 MSC) on the first day of life to the newborn FGR piglet improved damaged vasculature, restored the neurovascular unit, reduced brain inflammation and improved adverse neuronal and white matter changes present in the FGR newborn piglet brain. These findings could not be reproduced using MSCs alone. These results demonstrate cECFC treatment exerts beneficial effects on multiple cellular components in the FGR brain and may act as a neuroprotectant.

Hypoxia-ischemia in the immature rodent brain impairs serotonergic neuronal function in certain dorsal raphé nuclei.

Neonatal hypoxia-ischemia (HI) results in losses of serotonergic neurons in specific dorsal raphé nuclei. However, not all serotonergic raphé neurons are lost and it is therefore important to assess the function of remaining neurons in order to understand their potential to contribute to neurological disorders in the HI-affected neonate. The main objective of this study was to determine how serotonergic neurons, remaining in the dorsal raphé nuclei after neonatal HI, respond to an external stimulus (restraint stress). On postnatal day 3 (P3), male rat pups were randomly allocated to one of the following groups: (i) control + no restraint (n = 5), (ii) control + restraint (n = 6), (iii) P3 HI + no restraint (n = 5) or (iv) P3 HI + restraint (n = 7). In the two HI groups, rat pups underwent surgery to ligate the common carotid artery and were then exposed to 6% O2 for 30 minutes. Six weeks after P3 HI, on P45, rats were subjected to restraint stress for 30 minutes. Using dual immunolabeling for Fos protein, a marker for neuronal activity, and serotonin (5-hydroxytrypamine; 5-HT), numbers of Fos-positive 5-HT neurons were determined in five dorsal raphé nuclei. We found that restraint stress alone increased numbers of Fos-positive 5-HT neurons in all five dorsal raphé nuclei compared to control animals. However, following P3 HI, the number of stress-induced Fos-positive 5-HT neurons was decreased significantly in the dorsal raphé ventrolateral, interfascicular and ventral nuclei compared with control animals exposed to restraint stress. In contrast, numbers of stress-induced Fos-positive 5-HT neurons in the dorsal raphé dorsal and caudal nuclei were not affected by P3 HI. These data indicate that not only are dorsal raphé serotonergic neurons lost after neonatal HI, but also remaining dorsal raphé serotonergic neurons have reduced differential functional viability in response to an external stimulus. Procedures were approved by the University of Queensland Animal Ethics Committee (UQCCR958/08/NHMRC) on February 27, 2009.

Minocycline: a neuroprotective agent for hypoxic-ischemic brain injury in the neonate?

Minocycline is a second-generation tetracycline and a potential neuroprotective intervention following brain injury. However, despite the recognized beneficial effects of minocycline in a multitude of adult disease states, the clinical application of minocycline in neonates is contentious. Tetracyclines, as a class, are not usually administered to neonates, but there is compelling evidence that minocycline reduces brain injury after neonatal hypoxic-ischemic brain injury. This Review focuses on the evidence for minocycline use in neonates by considering aspects of pharmacology, drug regimens, functional outcomes, and mechanisms of action.

Ibuprofen Treatment Reduces the Neuroinflammatory Response and Associated Neuronal and White Matter Impairment in the Growth Restricted Newborn.

Intrauterine growth restriction (IUGR) is a condition where the fetus does not achieve optimal growth, commonly caused by placental insufficiency. The chronic decrease in blood flow restricts oxygen and nutrient supply to the fetus, which can damage numerous organ systems, with the fetal brain being particularly vulnerable. Although white matter and neuronal injury are evident in IUGR infants, the specific mechanisms underlying these changes are poorly understood. Inflammation is considered to be a main driver in exacerbating brain injury. Using a spontaneous piglet model of IUGR, we aim to determine whether administration of the anti-inflammatory drug ibuprofen will decrease inflammation at postnatal day 4 (P4). The treatment group received ibuprofen (20 mg/kg/day on day 1 and 10 mg/kg/day on days 2 and 3) in piglet formula during the morning feed each day and brains examined on P4. Markers of inflammation, apoptosis, cell proliferation, neuronal injury, and white matter injury were examined. Ibuprofen treatment ameliorated the increase in numbers of microglia and astrocytes in the parietal cortex and white matter tracts of the IUGR piglet brain on P4 as well as decreasing proinflammatory cytokines. Ibuprofen treatment prevented the reduction in apoptosis, neuronal cell counts, and myelin index in the IUGR piglets. Our findings demonstrate ibuprofen reduces the inflammatory response in the IUGR neonatal brain and concurrently reduces neuronal and white matter impairment.

Post-insult minocycline treatment attenuates hypoxia-ischemia-induced neuroinflammation and white matter injury in the neonatal rat: a comparison of two different dose regimens.

An increase in the number of activated microglia in the brain is a key feature of neuroinflammation after a hypoxic-ischemic insult to the preterm neonate and can contribute to white matter injury in the brain. Minocycline is a potent inhibitor of microglia and may have a role as a neuroprotective agent that ameliorates brain injury after hypoxia-ischemia in neonatal animal models. However to date large doses, pre-insult administration and short periods of treatment after hypoxia-ischemia have mostly been investigated in animal models making it difficult to translate minocycline's potential applicability to protect the human preterm neonatal brain exposed to hypoxia-ischemia. We investigated whether repeated doses of minocycline can minimize white matter injury and neuroinflammation one week after hypoxia-ischemia (right carotid artery ligation and 30 min 6% O(2)) in the post-natal day 3 rat pup. Two dosage regimens of minocycline were administered for one week; a high dose of 45 mg/kg 2h after hypoxia-ischemia then 22.5 mg/kg daily or a low dose 22.5 mg/kg 2h after hypoxia-ischemia then 10 mg/kg. Post-natal day 3 hypoxia-ischemia significantly reduced myelin content, numbers of O1- and O4-positive oligodendrocyte progenitor cells and increased activated microglia one week later on post-natal day 10. The low dose minocycline regimen was as effective as the high dose in ameliorating neuroinflammation after post-natal day 3 hypoxia-ischemia. However only the high dose regimen significantly attenuated reductions in O1- and O4-positive oligodendrocyte progenitor cells and myelin content. The low dose only significantly attenuated the reduction in O1-positive oligodendrocyte cell counts. Repeated, daily, post-insult treatment with minocycline abolished neuroinflammation and may provide neuroprotection to white matter for up to one week after hypoxia-ischemia in a rodent preterm model. The present findings suggest the potential clinical relevance of a repeated, daily minocycline treatment strategy, administered after a hypoxia-ischemia insult, as a therapeutic intervention for hypoxia-ischemia-affected preterm neonates.