The Temporal Relationship between Blood-Brain Barrier Integrity and Microglial Response following Neonatal Hypoxia Ischemia.

Blood-brain barrier (BBB) dysfunction and neuroinflammation are key mechanisms of brain injury. We performed a time-course study following neonatal hypoxia-ischemia (HI) to characterize these events. HI brain injury was induced in postnatal day 10 rats by single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). At 6, 12, 24, and 72 h (h) post-HI, brains were collected to assess neuropathology and BBB dysfunction. A significant breakdown of the BBB was observed in the HI injury group compared to the sham group from 6 h in the cortex and hippocampus (p < 0.001), including a significant increase in albumin extravasation (p < 0.0033) and decrease in basal lamina integrity and tight-junction proteins. There was a decrease in resting microglia (p < 0.0001) transitioning to an intermediate state from as early as 6 h post-HI, with the intermediate microglia peaking at 12 h (p < 0.0001), which significantly correlated to the peak of microbleeds. Neonatal HI insult leads to significant brain injury over the first 72 h that is mediated by BBB disruption within 6 h and a transitioning state of the resident microglia. Key BBB events coincide with the appearance of the intermediate microglial state and this relationship warrants further research and may be a key target for therapeutic intervention.

Phenotype of early-onset fetal growth restriction in sheep.

Introduction: Fetal growth restriction (FGR) is a common pregnancy complication, caused by placental insufficiency, with serious adverse consequences for development in utero and postnatal wellbeing. There are no antenatal treatments to improve growth or organ development in FGR, and animal models are essential to mimic the physiological adaptations in FGR and to assess potential interventions. This study aimed to identify the temporal nature of reduced developmental trajectory in fetuses with FGR, and to examine the effects of common factors that may mediate differential growth such as glucocorticoid treatment. We hypothesised that the trajectory of growth would be adversely impacted by FGR. Methods: FGR was induced via surgical placental insufficiency in fetal sheep (89 days gestation/0.6 gestation; n=135) and compared to age-matched controls over the last third of gestation and into neonatal life (n=153). Results: Body weight of FGR fetuses/lambs was significantly reduced compared to controls (p<0.0001) from 127 days of gestation (term is 148 days), with increased brain:body weight ratio (p<0.0001) indicative of brain sparing. All biometric measures of body size were reduced in the FGR group with the exception of biparietal (head) diameter. The trajectory of body growth in the last trimester of sheep pregnancy was significantly reduced in the FGR group compared to controls, and stillbirth rate increased with longer gestation. Discussion: This work provides a well characterised FGR animal model that mimics the known physiological adaptations in human pregnancy and can be used to determine the efficacy of potential interventions.

Perinatal compromise affects development, form, and function of the hippocampus part two; preclinical studies.

The hippocampus is a vital brain structure deep in the medial temporal lobe that mediates a range of functions encompassing emotional regulation, learning, memory, and cognition. Hippocampal development is exquisitely sensitive to perturbations and adverse conditions during pregnancy and at birth, including preterm birth, fetal growth restriction (FGR), acute hypoxic-ischaemic encephalopathy (HIE), and intrauterine inflammation. Disruptions to hippocampal development due to these conditions can have long-lasting functional impacts. Here, we discuss a range of preclinical models of prematurity and FGR and conditions that induce hypoxia and inflammation, which have been critical in elucidating the underlying mechanisms and cellular and subcellular structures implicated in hippocampal dysfunction. Finally, we discuss potential therapeutic targets to reduce the burden of these perinatal insults on the developing hippocampus. IMPACT: The review explores the preclinical literature examining the association between pregnancy and birth complications, and hippocampal form and function. The developmental processes and cellular mechanisms that are disrupted within the hippocampus following perinatal compromise are described, and potential therapeutic targets are discussed.

Perinatal compromise affects development, form, and function of the hippocampus part one; clinical studies.

The hippocampus is a neuron-rich specialised brain structure that plays a central role in the regulation of emotions, learning and memory, cognition, spatial navigation, and motivational processes. In human fetal development, hippocampal neurogenesis is principally complete by mid-gestation, with subsequent maturation comprising dendritogenesis and synaptogenesis in the third trimester of pregnancy and infancy. Dendritogenesis and synaptogenesis underpin connectivity. Hippocampal development is exquisitely sensitive to perturbations during pregnancy and at birth. Clinical investigations demonstrate that preterm birth, fetal growth restriction (FGR), and acute hypoxic-ischaemic encephalopathy (HIE) are common perinatal complications that alter hippocampal development. In turn, deficits in hippocampal development and structure mediate a range of neurodevelopmental disorders, including cognitive and learning problems, autism, and Attention-Deficit/Hyperactivity Disorder (ADHD). In this review, we summarise the developmental profile of the hippocampus during fetal and neonatal life and examine the hippocampal deficits observed following common human pregnancy complications. IMPACT: The review provides a comprehensive summary of the developmental profile of the hippocampus in normal fetal and neonatal life. We address a significant knowledge gap in paediatric research by providing a comprehensive summary of the relationship between pregnancy complications and subsequent hippocampal damage, shedding new light on this critical aspect of early neurodevelopment.

Neuroprotective effects of maternal melatonin administration in early-onset placental insufficiency and fetal growth restriction.

BACKGROUND: Early-onset fetal growth restriction (FGR) is associated with adverse outcomes. We hypothesised that maternal melatonin administration will improve fetal brain structure in FGR. METHODS: Surgery was performed on twin-bearing ewes at 88 days (0.6 gestation), and FGR induced in one twin via single umbilical artery ligation. Melatonin was administered intravenously (6 mg/day) to a group of ewes commencing on day of surgery until 127 days (0.85 gestation), when the ewe/fetuses were euthanized, and fetal brains collected. RESULTS: Study groups were control (n = 5), FGR (n = 5), control+melatonin (control+MLT; n = 6) and FGR+melatonin (FGR + MLT; n = 6). Melatonin administration did not significantly alter fetal body or brain weights. Myelin (CNPase+) fibre density was reduced in FGR vs. control animals in most brain regions examined (p < 0.05) and melatonin treatment restored CNPase fibre density. Similar but less pronounced effect was seen with mature myelin (MBP+) staining. Significant differences in activated microglia (Iba-1) activity were seen between lamb groups (MLT mitigated FGR effect) in periventricular white matter, subventricular zone and external capsule (p < 0.05). Similar effects were seen in astrogliosis (GFAP) in intragyral white matter and cortex. CONCLUSIONS: Maternal melatonin administration in early onset FGR led to improved myelination of white matter brain regions, possibly mediated by decreased inflammation. IMPACT: Maternal melatonin administration might lead to neuroprotection in the growth-restricted fetus, possibly via dampening neuroinflammation and enhancing myelination. This preclinical study adds to the body of work on this topic, and informs clinical translation. Neuroprotection likely to improve long-term outcomes of this vulnerable infant group.

Neuroprotective Action of Tacrolimus before and after Onset of Neonatal Hypoxic-Ischaemic Brain Injury in Rats.

(1) Background: Neonatal brain injury can lead to permanent neurodevelopmental impairments. Notably, suppressing inflammatory pathways may reduce damage. To determine the role of neuroinflammation in the progression of neonatal brain injury, we investigated the effect of treating neonatal rat pups with the immunosuppressant tacrolimus at two time points: before and after hypoxic-ischaemic (HI)-induced injury. (2) Methods: To induce HI injury, postnatal day (PND) 10 rat pups underwent single carotid artery ligation followed by hypoxia (8% oxygen, 90 min). Pups received daily tacrolimus (or a vehicle) starting either 3 days before HI on PND 7 (pre-HI), or 12 h after HI (post-HI). Four doses were tested: 0.025, 0.05, 0.1 or 0.25 mg/kg/day. Pups were euthanised at PND 17 or PND 50. (3) Results: All tacrolimus doses administered pre-HI significantly reduced brain infarct size and neuronal loss, increased the number of resting microglia and reduced cellular apoptosis (p < 0.05 compared to control). In contrast, only the highest dose of tacrolimus administered post-HI (0.25 mg/kg/day) reduced brain infarct size (p < 0.05). All doses of tacrolimus reduced pup weight compared to the controls. (4) Conclusions: Tacrolimus administration 3 days pre-HI was neuroprotective, likely mediated through neuroinflammatory and cell death pathways. Tacrolimus post-HI may have limited capacity to reduce brain injury, with higher doses increasing rat pup mortality. This work highlights the benefits of targeting neuroinflammation during the acute injurious period. More specific targeting of neuroinflammation, e.g., via T-cells, warrants further investigation.

Cardiovascular responses to mild perinatal asphyxia in growth-restricted preterm lambs.

Growth-restricted neonates have worse outcomes after perinatal asphyxia, with more severe metabolic acidosis than appropriately grown neonates. The cardiovascular physiology associated with fetal growth restriction (FGR) may alter their response to asphyxia. However, research on asphyxia in FGR is limited. Here we compared cardiovascular hemodynamics in preterm FGR and control lambs during mild perinatal asphyxia. We induced FGR in one twin at 89 days gestation (term 148 days), while the other served as a control. At 126 days gestation, lambs were instrumented to allow arterial blood pressure and regional blood flow recording, and then mild perinatal asphyxia was induced by umbilical cord clamping, and resuscitation followed neonatal guidelines. FGR lambs maintained carotid blood flow (CBF) for 7 min, while control lambs rapidly decreased CBF (P < 0.05). Fewer growth-restricted lambs needed chest compressions for return of spontaneous circulation (ROSC) (17 vs. 83%, P = 0.02). The extent of blood pressure overshoot after ROSC was similar, but it took longer for MAP to return to baseline in FGR lambs (18.83 ± 0.00 vs. 47.67 ± 0.00 min, P = 0.003). Growth-restricted lambs had higher CBF after ROSC (P < 0.05) and displayed CBF overshoot, unlike control lambs (P < 0.03). In conclusion, preterm growth-restricted lambs show resilience during perinatal asphyxia based on prolonged CBF maintenance and reduced need for chest compressions during resuscitation. However, CBF overshoot after ROSC may increase the risk of cerebrovascular injury in FGR.NEW & NOTEWORTHY Preterm growth-restricted lambs maintain carotid blood flow for longer than control lambs during asphyxia and have a lower requirement for chest compressions than control lambs during resuscitation. Preterm growth-restricted, but not control, lambs displayed an overshoot in carotid blood flow following return of spontaneous circulation.

Cardiovascular decline in offspring during the perinatal period in an ovine model of fetal growth restriction.

Fetal growth restriction (FGR) increases the risk cardiovascular disease (CVD) in adulthood. Placental insufficiency and subsequent chronic fetal hypoxemia are causal factors for FGR, leading to a redistribution of blood flow that prioritizes vital organs. Subclinical signs of cardiovascular dysfunction are evident in growth-restricted neonates; however, the mechanisms programming for CVD in adulthood remain unknown. This study aimed to determine the potential mechanisms underlying structural and functional changes within the heart and essential (carotid) and nonessential (femoral) vascular beds in growth-restricted lambs. Placental insufficiency was surgically induced in ewes at 89 days gestational age (dGA, term = 148dGA). Three age groups were investigated: fetal (126dGA), newborn (24 h after preterm birth), and 4-wk-old lambs. In vivo and histological assessments of cardiovascular indices were undertaken. Resistance femoral artery function was assessed via in vitro wire myography and blockade of key vasoactive pathways including nitric oxide, prostanoids, and endothelium-dependent hyperpolarization. All lambs were normotensive throughout the first 4 wk of life. Overall, the FGR cohort had more globular hearts compared with controls (P = 0.0374). A progressive decline in endothelium-dependent vasodilation was demonstrated in FGR lambs compared with controls. Further investigation revealed that impairment of the prostanoid pathway may drive this reduction in vasodilatory capacity. Clinical indicators of CVD were not observed in our FGR lambs. However, subclinical signs of cardiovascular dysfunction were present in our FGR offspring. This study provides insight into potential mechanisms, such as the prostanoid pathway, that may warrant therapeutic interventions to improve cardiovascular development in growth-restricted newborns.NEW & NOTEWORTHY Our findings provide novel insight into the potential mechanisms that program for cardiovascular dysfunction in growth-restricted neonates as our growth-restricted lambs exhibited a progressive decline in endothelium-dependent vasodilation in the femoral artery between birth and 4 wk of age. Subsequent analyses indicated that this reduction in vasodilatory capacity is likely to be mediated by the prostanoid pathway and prostanoids could be a potential target for therapeutic interventions for fetal growth restriction (FGR).

The medullary serotonergic centres involved in cardiorespiratory control are disrupted by fetal growth restriction.

Fetal growth restriction (FGR) is associated with cardiovascular and respiratory complications after birth and beyond. Despite research showing a range of neurological changes following FGR, little is known about how FGR affects the brainstem cardiorespiratory control centres. The primary neurons that release serotonin reside in the brainstem cardiorespiratory control centres and may be affected by FGR. At two time points in the last trimester of sheep brain development, 110 and 127 days of gestation (0.74 and 0.86 of gestation), we assessed histopathological alterations in the brainstem cardiorespiratory control centres of the pons and medulla in early-onset FGR versus control fetal sheep. The FGR cohort were hypoxaemic and asymmetrically growth restricted. Compared to the controls, the brainstem of FGR fetuses exhibited signs of neuropathology, including elevated cell death and reduced cell proliferation, grey and white matter deficits, and evidence of oxidative stress and neuroinflammation. FGR brainstem pathology was predominantly observed in the medullary raphé nuclei, hypoglossal nucleus, nucleus ambiguous, solitary tract and nucleus of the solitary tract. The FGR groups showed imbalanced brainstem serotonin and serotonin 1A receptor abundance in the medullary raphé nuclei, despite evidence of increased serotonin staining within vascular regions of placentomes collected from FGR fetuses. Our findings demonstrate both early and adaptive brainstem neuropathology in response to placental insufficiency. KEY POINTS: Early-onset fetal growth restriction (FGR) was induced in fetal sheep, resulting in chronic fetal hypoxaemia. Growth-restricted fetuses exhibit persistent neuropathology in brainstem nuclei, characterised by disrupted cell proliferation and reduced neuronal cell number within critical centres responsible for the regulation of cardiovascular and respiratory functions. Elevated brainstem inflammation and oxidative stress suggest potential mechanisms contributing to the observed neuropathological changes. Both placental and brainstem levels of 5-HT were found to be impaired following FGR.

Endothelial colony forming cell administration promotes neurovascular unit development in growth restricted and appropriately grown fetal lambs.

BACKGROUND: Fetal growth restriction (FGR) is associated with deficits in the developing brain, including neurovascular unit (NVU) dysfunction. Endothelial colony forming cells (ECFC) can mediate improved vascular stability, and have demonstrated potential to enhance vascular development and protection. This investigation examined whether ECFCs from human umbilical cord blood (UCB) enhanced NVU development in FGR and appropriate for gestational age (AGA) fetal sheep. METHODS: Twin-bearing ewes had surgery performed at 88-90 days' gestation, inducing FGR in one fetus. At 113 days, ECFCs (1 × 107 cells) cultured from human UCB were administered intravenously to fetal sheep in utero. At 127 days, ewes and their fetuses were euthanised, fetal brains collected, and NVU components analysed by immunohistochemistry. RESULTS: Twenty-four fetal lambs, arranged in four groups: AGA (n = 7), FGR (n = 5), AGA + ECFC (n = 6), and FGR + ECFC (n = 6), were included in analyses. FGR resulted in lower body weight than AGA (P = 0.002) with higher brain/body weight ratio (P = 0.003). ECFC treatment was associated with increased vascular density throughout the brain in both AGA + ECFC and FGR + ECFC groups, as well as increased vascular-astrocyte coverage and VEGF expression in the cortex (P = 0.003, P = 0.0006, respectively) and in the subcortical white matter (P = 0.01, P = 0.0002, respectively) when compared with the untreated groups. CONCLUSIONS: ECFC administration enhanced development of NVU components in both the AGA and FGR fetal brain. Further investigation is required to assess how to optimise the enhanced angiogenic capabilities of ECFCs to provide a therapeutic strategy to protect the developing NVU against vulnerabilities associated with FGR.

Ganaxolone versus Phenobarbital for Neonatal Seizure Management.

OBJECTIVE: Seizures are more common in the neonatal period than at any other stage of life. Phenobarbital is the first-line treatment for neonatal seizures and is at best effective in approximately 50% of babies, but may contribute to neuronal injury. Here, we assessed the efficacy of phenobarbital versus the synthetic neurosteroid, ganaxolone, to moderate seizure activity and neuropathology in neonatal lambs exposed to perinatal asphyxia. METHODS: Asphyxia was induced via umbilical cord occlusion in term lambs at birth. Lambs were treated with ganaxolone (5mg/kg/bolus then 5mg/kg/day for 2 days) or phenobarbital (20mg/kg/bolus then 5mg/kg/day for 2 days) at 6 hours. Abnormal brain activity was classified as stereotypic evolving (SE) seizures, epileptiform discharges (EDs), and epileptiform transients (ETs) using continuous amplitude-integrated electroencephalographic recordings. At 48 hours, lambs were euthanized for brain pathology. RESULTS: Asphyxia caused abnormal brain activity, including SE seizures that peaked at 18 to 20 hours, EDs, and ETs, and induced neuronal degeneration and neuroinflammation. Ganaxolone treatment was associated with an 86.4% reduction in the number of seizures compared to the asphyxia group. The total seizure duration in the asphyxia+ganaxolone group was less than the untreated asphyxia group. There was no difference in the number of SE seizures between the asphyxia and asphyxia+phenobarbital groups or duration of SE seizures. Ganaxolone treatment, but not phenobarbital, reduced neuronal degeneration within hippocampal CA1 and CA3 regions, and cortical neurons, and ganaxolone reduced neuroinflammation within the thalamus. INTERPRETATION: Ganaxolone provided better seizure control than phenobarbital in this perinatal asphyxia model and was neuroprotective for the newborn brain, affording a new therapeutic opportunity for treatment of neonatal seizures. ANN NEUROL 2022;92:1066-1079.

An Optimized and Detailed Step-by-Step Protocol for the Analysis of Neuronal Morphology in Golgi-Stained Fetal Sheep Brain.

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure gray matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi-staining protocols have been optimized, and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi-stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semiautomated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking, and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) of gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (µm), branch number, branch order, and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density, and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labor-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

Altered trajectory of neurodevelopment associated with fetal growth restriction.

Fetal growth restriction (FGR) is principally caused by suboptimal placental function. Poor placental function causes an under supply of nutrients and oxygen to the developing fetus, restricting development of individual organs and overall growth. Estimated fetal weight below the 10th or 3rd percentile with uteroplacental dysfunction, and knowledge regarding the onset of growth restriction (early or late), provide diagnostic criteria for fetuses at greatest risk for adverse outcome. Brain development and function is altered with FGR, with ongoing clinical and preclinical studies elucidating neuropathological etiology. During the third trimester of pregnancy, from ~28 weeks gestation, neurogenesis is complete and neuronal complexity is expanding, through axonal and dendritic outgrowth, dendritic branching and synaptogenesis, accompanied by myelin production. Fetal compromise over this period, as occurs in FGR, has detrimental effects on these processes. Total brain volume and grey matter volume is reduced in infants with FGR, first evident in utero, with cortical volume particularly vulnerable. Imaging studies show that cerebral morphology is disturbed in FGR, with altered cerebral cortex, volume and organization of brain networks, and reduced connectivity of long- and short-range circuits. Thus, FGR induces a deviation in brain development trajectory affecting both grey and white matter, however grey matter volume is preferentially reduced, contributed by cell loss, and reduced neurite outgrowth of surviving neurons. In turn, cell-to-cell local networks are adversely affected in FGR, and whole brain left and right intrahemispheric connections and interhemispheric connections are altered. Importantly, disruptions to region-specific brain networks are linked to cognitive and behavioral impairments.

Umbilical cord blood therapy modulates neonatal hypoxic ischemic brain injury in both females and males.

Preclinical and clinical studies have shown that sex is a significant risk factor for perinatal morbidity and mortality, with males being more susceptible to neonatal hypoxic ischemic (HI) brain injury. No study has investigated sexual dimorphism in the efficacy of umbilical cord blood (UCB) cell therapy. HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received 3 doses of UCB cells (PND11, 13, 20) and underwent behavioural testing. On PND50, brains were collected for immunohistochemical analysis. Behavioural and neuropathological outcomes were assessed for sex differences. HI brain injury resulted in a significant decrease in brain weight and increase in tissue loss in females and males. Females and males also exhibited significant cell death, region-specific neuron loss and long-term behavioural deficits. Females had significantly smaller brains overall compared to males and males had significantly reduced neuron numbers in the cortex compared to females. UCB administration improved multiple aspects of neuropathology and functional outcomes in males and females. Females and males both exhibited injury following HI. This is the first preclinical evidence that UCB is an appropriate treatment for neonatal brain injury in both female and male neonates.

Cardiovascular and Cerebrovascular Implications of Growth Restriction: Mechanisms and Potential Treatments.

Fetal growth restriction (FGR) is a common complication of pregnancy, resulting in a fetus that fails to reach its genetically determined growth potential. Whilst the fetal cardiovascular response to acute hypoxia is well established, the fetal defence to chronic hypoxia is not well understood due to experiment constraints. Growth restriction results primarily from reduced oxygen and nutrient supply to the developing fetus, resulting in chronic hypoxia. The fetus adapts to chronic hypoxia by redistributing cardiac output via brain sparing in an attempt to preserve function in the developing brain. This review highlights the impact of brain sparing on the developing fetal cardiovascular and cerebrovascular systems, as well as emerging long-term effects in offspring that were growth restricted at birth. Here, we explore the pathogenesis associated with brain sparing within the cerebrovascular system. An increased understanding of the mechanistic pathways will be critical to preventing neuropathological outcomes, including motor dysfunction such as cerebral palsy, or behaviour dysfunctions including autism and attention-deficit/hyperactivity disorder (ADHD).

Melatonin augments the neuroprotective effects of hypothermia in lambs following perinatal asphyxia.

Therapeutic hypothermia (TH) is standard care in high-resource birth settings for infants with neonatal encephalopathy. TH is partially effective and adjuvant therapies are needed. Here, we examined whether the antioxidant melatonin (MLT) provides additive benefit with TH, compared to TH alone or MLT alone, to improve recovery from acute encephalopathy in newborn lambs. Immediately before cesarean section delivery, we induced asphyxia in fetal sheep via umbilical cord occlusion until mean arterial blood pressure fell from 55 ± 3 mm Hg in sham controls to 18-20 mm Hg (10.1 ± 1.5 minutes). Lambs were delivered and randomized to control, control + MLT (60 mg iv, from 30 minutes to 24 hours), asphyxia, asphyxia + TH (whole-body cooling to 35.1 ± 0.8°C vs. 38.3 ± 0.17°C in sham controls, from 4-28 hours), asphyxia + MLT, and asphyxia + TH + MLT. At 72 hours, magnetic resonance spectroscopy (MRS) was undertaken, and then brains were collected for neuropathology assessment. Asphyxia induced abnormal brain metabolism on MRS with increased Lactate:NAA (P = .003) and reduced NAA:Choline (P = .005), induced apoptotic and necrotic cell death across gray and white matter brain regions (P < .05), and increased neuroinflammation and oxidative stress (P < .05). TH and MLT were independently associated with region-specific reductions in oxidative stress, inflammation, and cell death, compared to asphyxia alone. There was an interaction between TH and MLT such that the NAA:Choline ratio was not significantly different after asphyxia + TH + MLT compared to sham controls but had a greater overall reduction in neuropathology than either treatment alone. This study demonstrates that, in newborn lambs, combined TH + MLT for neonatal encephalopathy provides significantly greater neuroprotection than either alone. These results will guide the development of further trials for neonatal encephalopathy.

Optimization of behavioral testing in a long-term rat model of hypoxic ischemic brain injury.

BACKGROUND: Hypoxic ischemic (HI) brain injury is a significant cause of childhood neurological deficits. Preclinical rodent models are often used to study these deficits; however, no preclinical study has determined which behavioral tests are most appropriate for long-term follow up after neonatal HI. METHODS: HI brain injury was induced in postnatal day (PND) 10 rat pups using the Rice-Vannucci method of unilateral carotid artery ligation. Rats underwent long-term behavioral testing to assess motor and cognitive outcomes between PND11-50. Behavioral scores were transformed into Z-scores and combined to create composite behavioral scores. RESULTS: HI rats showed a significant deficit in three out of eight behavioral tests: negative geotaxis analysis, the cylinder test and the novel object recognition test. These individual test outcomes were transformed into Z-scores and combined to create a composite Z-score. This composite z-score showed that HI rats had a significantly increased behavioral burden over the course of the experiment. CONCLUSION: In this study we have identified tests that highlight specific cognitive and motor deficits in a rat model of neonatal HI. Due to the high variability in this model of neonatal HI brain injury, significant impairment is not always observed in individual behavioral tests, but by combining outcomes from these individual tests, long-term behavioral burden can be measured.

Multiple doses of umbilical cord blood cells improve long-term brain injury in the neonatal rat.

BACKGROUND: Hypoxic ischemic (HI) insults during pregnancy and birth can result in neurodevelopmental disorders, such as cerebral palsy. We have previously shown that a single dose of umbilical cord blood (UCB) cells is effective at reducing short-term neuroinflammation and improves short and long-term behavioural outcomes in rat pups. A single dose of UCB was not able to modulate long-term neuroinflammation or brain tissue loss. In this study we examined whether multiple doses of UCB can modulate neuroinflammation, decrease cerebral tissue damage and improve behavioural outcomes when followed up long-term. METHODS: HI injury was induced in postnatal day 10 (PND10) rat pups using the Rice-Vannucci method of carotid artery ligation. Pups received either 1 dose (PND11), or 3 doses (PND11, 13, 20) of UCB cells. Rats were followed with behavioural testing, to assess both motor and cognitive outcomes. On PND50, brains were collected for analysis. RESULTS: HI brain injury in rat pups caused significant behavioural deficits. These deficits were significantly improved by multiple doses of UCB. HI injury resulted in a significant decrease in brain weight and left hemisphere tissue, which was improved by multiple doses of UCB. HI resulted in increased cerebral apoptosis, loss of neurons and upregulation of activated microglia. Multiple doses of UCB modulated these neuropathologies. A single dose of UCB at PND11 did not improve behavioural or neuropathological outcomes. CONCLUSIONS: Treatment with repeated doses of UCB is more effective than a single dose for reducing tissue damage, improving brain pathology and restoring behavioural deficits following perinatal brain injury.

Does Antenatal Betamethasone Alter White Matter Brain Development in Growth Restricted Fetal Sheep?

Fetal growth restriction (FGR) is a common complication of pregnancy often associated with neurological impairments. Currently, there is no treatment for FGR, hence it is likely these babies will be delivered prematurely, thus being exposed to antenatal glucocorticoids. While there is no doubt that antenatal glucocorticoids reduce neonatal mortality and morbidities, their effects on the fetal brain, particularly in FGR babies, are less well recognized. We investigated the effects of both short- and long-term exposure to antenatal betamethasone treatment in both FGR and appropriately grown fetal sheep brains. Surgery was performed on pregnant Border-Leicester Merino crossbred ewes at 105-110 days gestation (term ~150 days) to induce FGR by single umbilical artery ligation (SUAL) or sham surgery. Ewes were then treated with a clinical dose of betamethasone (11.4 mg intramuscularly) or saline at 113 and 114 days gestation. Animals were euthanized at 115 days (48 h following the initial betamethasone administration) or 125 days (10 days following the initial dose of betamethasone) and fetal brains collected for analysis. FGR fetuses were significantly smaller than controls (115 days: 1.68 ± 0.11 kg vs. 1.99 ± 0.11 kg, 125 days: 2.70 ± 0.15 kg vs. 3.31 ± 0.20 kg, P < 0.001) and betamethasone treatment reduced body weight in both control (115 days: 1.64 ± 0.10 kg, 125 days: 2.53 ± 0.10 kg) and FGR fetuses (115 days: 1.41 ± 0.10 kg, 125 days: 2.16 ± 0.17 kg, P < 0.001). Brain: body weight ratios were significantly increased with FGR (P < 0.001) and betamethasone treatment (P = 0.002). Within the fetal brain, FGR reduced CNPase-positive myelin staining in the subcortical white matter (SCWM; P = 0.01) and corpus callosum (CC; P = 0.01), increased GFAP staining in the SCWM (P = 0.02) and reduced the number of Olig2 cells in the periventricular white matter (PVWM; P = 0.04). Betamethasone treatment significantly increased CNPase staining in the external capsule (EC; P = 0.02), reduced GFAP staining in the CC (P = 0.03) and increased Olig2 staining in the SCWM (P = 0.04). Here we show that FGR has progressive adverse effects on the fetal brain, particularly within the white matter. Betamethasone exacerbated growth restriction in the FGR offspring, but betamethasone did not worsen white matter brain injury.

Is Umbilical Cord Blood Therapy an Effective Treatment for Early Lung Injury in Growth Restriction?

Fetal growth restriction (FGR) and prematurity are often co-morbidities, and both are risk factors for lung disease. Despite advances in early delivery combined with supportive ventilation, rates of ventilation-induced lung injury (VILI) remain high. There are currently no protective treatments or interventions available that target lung morbidities associated with FGR preterm infants. Stem cell therapy, such as umbilical cord blood (UCB) cell administration, demonstrates an ability to attenuate inflammation and injury associated with VILI in preterm appropriately grown animals. However, no studies have looked at the effects of stem cell therapy in growth restricted newborns. We aimed to determine if UCB treatment could attenuate acute inflammation in the first 24 h of ventilation, comparing effects in lambs born preterm following FGR with those born preterm but appropriately grown (AG). Placental insufficiency (FGR) was induced by single umbilical artery ligation in twin-bearing ewes at 88 days gestation, with twins used as control (appropriately grown, AG). Lambs were delivered preterm at ~126 days gestation (term is 150 days) and randomized to either immediate euthanasia (unventilated controls, AGUVC and FGRUVC) or commenced on 24 h of gentle supportive ventilation (AGV and FGRV) with additional cohorts receiving UCB treatment at 1 h (AGCELLS, FGRCELLS). Lungs were collected at post-mortem for histological and biochemical examination. Ventilation caused lung injury in AG lambs, as indicated by decreased septal crests and elastin density, as well as increased inflammation. Lung injury in AG lambs was attenuated with UCB therapy. Ventilated FGR lambs also sustained lung injury, albeit with different indices compared to AG lambs; in FGR, ventilation reduced septal crest density, reduced alpha smooth muscle actin density and reduced cell proliferation. UCB treatment in ventilated FGR lambs further decreased septal crest density and increased collagen deposition, however, it increased angiogenesis as evidenced by increased vascular endothelial growth factor (VEGF) expression and vessel density. This is the first time that a cell therapy has been investigated in the lungs of growth restricted animals. We show that the uterine environment can alter the response to both secondary stress (ventilation) and therapy (UCB). This study highlights the need for further research on the potential impact of novel therapies on a growth restricted offspring.