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BACKGROUND: Neonatal hypoxic-ischemic encephalopathy disproportionately affects low- and middle-income countries, where ≈96% of affected infants reside. The current standard of care, therapeutic hypothermia, is frequently ineffective in this setting, likely because injury may be occurring earlier during labor. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal caffeine administration in near-term lambs following global ischemic injury to support the development of earlier treatment strategies targeting the fetus in utero as well as the infant postnatally. METHODS: Ewes were randomly assigned to receive either 1 g IV caffeine citrate or placebo before delivery and placental transport assessed. Near-term lambs (141-143 days) of both sexes were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Lambs that received caffeine in utero also received 20 mg/kg IV caffeine citrate following resuscitation and 10 mg/(kg·d) IV for 2 days. An additional cohort received 60 mg/kg followed by 30 mg/(kg·d) (low dose versus high dose) postnatally. Biochemical, histological, and neurological outcome measures in lambs were assessed over a 6-day period. RESULTS: Perinatal caffeine administration demonstrated excellent placental transport kinetics and was well tolerated with lamb plasma levels comparable to those targeted in neonates with apnea of prematurity. Caffeine administration resulted in a systemic immunomodulatory effect, evidenced by significant reductions in proinflammatory IP-10 levels. Treated lambs demonstrated improved neurodevelopmental outcomes, while histological analysis revealed that caffeine reduced gray matter injury and attenuated inflammation in the cingulate and parasagittal cortex. This neuroprotective effect was greater and via a different mode of action than we previously reported for azithromycin. A higher caffeine dosing regimen demonstrated significant toxicity. CONCLUSIONS: Perinatal caffeine administration is well tolerated, attenuates systemic and brain inflammation, and contributes to improvements in histological and neurological outcomes in an ovine model of neonatal hypoxic-ischemic encephalopathy.

This Viewpoint describes the advent of neonatal neurocritical care training, the data supporting the need and its efficacy, and the ways this training can optimize outcomes for vulnerable patients and their families.

Hypoxic-ischemic encephalopathy (HIE) is a common neurological syndrome in newborns with high mortality and morbidity. Therapeutic hypothermia (TH), which is standard of care for HIE, mitigates brain injury by suppressing anaerobic metabolism. However, more than 40% of HIE neonates have a poor outcome, even after TH. This study aims to provide metabolic biomarkers for predicting the outcomes of hypoxia-ischemia (HI) after TH using hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy. Postnatal day 10 (P10) mice with HI underwent TH at 1 h and were scanned at 6-8 h (P10), 24 h (P11), 7 days (P17), and 21 days (P31) post-HI on a 14.1-T NMR spectrometer. The metabolic images were collected, and the conversion rate from pyruvate to lactate and the ratio of lactate to pyruvate in the injured left hemisphere (kPL(L) and Lac/Pyr(L), respectively) were calculated at each timepoint. The outcomes of TH were determined by the assessments of brain injury on T2-weighted images and behavioral tests at later timepoint. kPL(L) and Lac/Pyr(L) over time between the good-outcome and poor-outcome groups and across timepoints within groups were analyzed. We found significant differences in temporal trends of kPL(L) and Lac/Pyr(L) between groups. In the good-outcome group, kPL(L) increased until P31 with a significantly higher value at P31 compared with that at P10, while the level of Lac/Pyr(L) at P31 was notably higher than those at all other timepoints. In the poor-outcome group, kPL(L) and Lac/Pyr(L) increased within 24 h. The kPL(L) value at P11 was considerably higher compared with P10. Discrete temporal changes of kPL(L) and Lac/Pyr(L) after TH between the good-outcome and poor-outcome groups were seen as early as 24 h after HI, reflecting various TH effects on brain anaerobic metabolism, which may provide insights for early screening for response to TH.

Recent insights into the frequency of occurrence and the genetic and mechanistic basis of nervous system disease have demonstrated that neurologic disorders occur as a spectrum across all ages. To meet future needs of patients with neurologic disease of all ages and prepare for increasing implementaton of precision therapies, greater integration of child and adult neurology residency training is needed. ANN NEUROL 2023;94:1005-1007.

BackgroundHypoxic-ischemic brain injury/encephalopathy affects about 1.15 million neonates per year, 96% of whom are born in low- and middle-income countries. Therapeutic hypothermia is not effective in this setting, possibly because injury occurs significantly before birth. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal azithromycin administration in near-term lambs following global ischemic injury to support earlier treatment approaches.MethodsEwes and their lambs of both sexes (n=34, 141-143 days) were randomly assigned to receive azithromycin or placebo before delivery as well as postnatally. Lambs were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Outcomes were assessed over a 6-day period.ResultsWhile maternal azithromycin exhibited relatively low placental transfer, azithromycin-treated lambs recovered spontaneous circulation faster following the initiation of cardiopulmonary resuscitation and were extubated sooner. Additionally, peri- and postnatal azithromycin administration was well tolerated, demonstrating a 77-hour plasma elimination half-life, as well as significant accumulation in the brain and other tissues. Azithromycin administration resulted in a systemic immunomodulatory effect, demonstrated by reductions in proinflammatory IL-6 (interleukin-6) levels. Treated lambs exhibited a trend toward improved neurodevelopmental outcomes while histological analysis revealed that azithromycin supported white matter preservation and attenuated inflammation in the cingulate and parasagittal cortex.ConclusionsPerinatal azithromycin administration enhances neonatal resuscitation, attenuates neuroinflammation, and supports limited improvement of select histological outcomes in an ovine model of hypoxic-ischemic brain injury/encephalopathy.

Originally approved by the U.S. Food and Drug Administration (FDA) for its antihistamine properties, clemastine can also promote white matter integrity and has shown promise in the treatment of demyelinating diseases such as multiple sclerosis. Here, we conducted an in-depth analysis of the feasibility, safety, and neuroprotective efficacy of clemastine administration in near-term lambs (n = 25, 141-143 days) following a global ischemic insult induced via an umbilical cord occlusion (UCO) model. Lambs were randomly assigned to receive clemastine or placebo postnatally, and outcomes were assessed over a six-day period. Clemastine administration was well tolerated. While treated lambs demonstrated improvements in inflammatory scores, their neurodevelopmental outcomes were unchanged.

The developing brain is uniquely susceptible to oxidative stress and endogenous antioxidant mechanisms are not sufficient to prevent injury from a hypoxic-ischemic challenge. Glutathione peroxidase (GPX1) activity reduces hypoxic-ischemic injury. Therapeutic hypothermia also reduces hypoxic-ischemic injury, in the rodent and the human brain, but the benefit is limited. Here, we combined GPX1 overexpression with hypothermia in a P9 mouse model of hypoxia-ischemia (HI) to test the effectiveness of both treatments together. Histological analysis showed that WT mice with hypothermia were less injured than WT with normothermia. In the GPX1-tg mice, however, despite a lower median score in the hypothermia treated mice, there was no significant difference between hypothermia and normothermia. GPX1 protein expression was higher in the cortex of all transgenic groups at 30 min and 24 h, as well as in WT 30 min after HI, with and without hypothermia. GPX1 was higher in the hippocampus of all transgenic groups and WT with HI and normothermia, at 24 h, but not at 30 min. Spectrin 150 was higher in all groups with HI, while spectrin 120 was higher in HI groups only at 24 h. There was reduced ERK1/2 activation in both WT and GPX1-tg HI at 30 min. Thus, with a relatively moderate insult we see a benefit with cooling in the WT, but not the GPX1-tg mouse brain. The fact that we see no benefit with increased GPx1 here in the P9 model (unlike in the P7 model), may indicate that oxidative stress in these older mice is elevated to an extent that increased GPx1 is insufficient for reducing injury. The lack of benefit of overexpressing GPX1 in conjunction with hypothermia after HI indicates that pathways triggered by GPX1 overexpression may interfere with the neuroprotective mechanisms provided by HT.

BackgroundWe previously reported that increasing severity of watershed (WS) injury in neonatal magnetic resonance imaging (MRI) is associated with worse language outcomes in early childhood. In the present study, we investigated the relationship between neonatal injury patterns and cognitive profile in adolescents with neonatal encephalopathy.MethodsTerm neonates with encephalopathy were prospectively enrolled and imaged using brain MRI from 1999 to 2008. Neonatal brain injury was scored according to the degree of injury in WS and basal ganglia/thalamus (BG/T) areas. The children underwent a neurocognitive assessment and follow-up brain MRI at the age of 10-16 years. The relationship between neonatal brain injury patterns and adolescent cognitive outcomes was assessed.ResultsIn a cohort of 16 children, neonatal MRI showed WS injury in 7, BG/T injury in 2, and normal imaging in 7. Children with WS injury had lower estimated overall cognitive ability than those with normal imaging. Increasing WS injury score was associated with decreasing estimated overall cognitive ability, Perceptual Reasoning Index, and digit span score.ConclusionsChildren with the WS injury are at an increased risk of having problems in long-term intellectual ability. These cognitive outcomes may underlie early language difficulties seen in children with neonatal WS injury.ImpactAdolescents with a history of neonatal encephalopathy and watershed pattern of injury on neonatal brain magnetic resonance imaging (MRI) had lower overall cognitive ability, perceptual reasoning skills, and auditory working memory than those with normal neonatal imaging. Children with post-neonatal epilepsy and cerebral palsy had the worst cognitive outcomes. Watershed pattern of injury confers high long-term differences in intellectual ability.

The central nervous system (CNS) is enriched with important classes of lipids, in which cholesterol is known to make up a major portion of myelin sheaths, besides being a structural and functional unit of CNS cell membranes. Unlike in the adult brain, where the cholesterol pool is relatively stable, cholesterol is synthesized and accumulated at the highest rate in the developing brain to meet the needs of rapid brain growth at this stage, which is also a critical period for neuroplasticity. In addition to its biophysical role in membrane organization, cholesterol is crucial for brain development due to its involvement in brain patterning, myelination, neuronal differentiation, and synaptogenesis. Thus any injuries to the immature brain that affect cholesterol homeostasis may have long-term adverse neurological consequences. In this review, we describe the unique features of brain cholesterol biosynthesis and metabolism, cholesterol trafficking between different cell types, and highlight cholesterol-dependent biological processes during brain maturation. We also discuss the association of impaired cholesterol homeostasis with several forms of perinatal brain disorders in term and preterm newborns, including hypoxic-ischemic encephalopathy. Strategies targeting the cholesterol pathways may open new avenues for the diagnosis and treatment of developmental brain injury.

Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal morbidity and mortality worldwide. Approximately 1 million infants born with HIE each year survive with cerebral palsy and/or serious cognitive disabilities. While infants born with mild and severe HIE frequently result in predictable outcomes, infants born with moderate HIE exhibit variable outcomes that are highly unpredictable. Here, we describe an umbilical cord occlusion (UCO) model of moderate HIE with a 6-day follow-up. Near-term lambs (n = 27) were resuscitated after the induction of 5 min of asystole. Following recovery, lambs were assessed to define neurodevelopmental outcomes. At the end of this period, lambs were euthanized, and brains were harvested for histological analysis. Compared with prior models that typically follow lambs for 3 days, the observation of neurobehavioral outcomes for 6 days enabled identification of animals that recover significant neurological function. Approximately 35% of lambs exhibited severe motor deficits throughout the entirety of the 6-day course and, in the most severely affected lambs, developed spastic diparesis similar to that observed in infants who survive severe neonatal HIE (severe, UCOs). Importantly, and similar to outcomes in human neonates, while initially developing significant acidosis and encephalopathy, the remainder of the lambs in this model recovered normal motor activity and exhibited normal neurodevelopmental outcomes by 6 days of life (improved, UCOi). The UCOs group exhibited gliosis and inflammation in both white and gray matters, oligodendrocyte loss, neuronal loss, and cellular death in the hippocampus and cingulate cortex. While the UCOi group exhibited more cellular death and gliosis in the parasagittal cortex, they demonstrated more preserved white matter markers, along with reduced markers of inflammation and lower cellular death and neuronal loss in Ca3 of the hippocampus compared with UCOs lambs. Our large animal model of moderate HIE with prolonged follow-up will help further define pathophysiologic drivers of brain injury while enabling identification of predictive biomarkers that correlate with disease outcomes and ultimately help support development of therapeutic approaches to this challenging clinical scenario.

The cellular responses to hypoxia or hypoxia-ischemia (HI) are governed largely by the hypoxia-inducible factor (HIF) family of transcription factors. Our previous studies show that HIF-1α induction is an important factor that mediates protective effects in the brain after neonatal HI. In the present study, we investigated the contribution of another closely related HIF α isoform, HIF-2α, specifically the neuronal HIF-2α, to brain HI injury. Homozygous transgenic mice with a floxed exon 2 of HIF-2α were bred with CaMKIIα-Cre mice to generate a mouse line with selective deletion of HIF-2α in forebrain neurons. These mice, along with their wildtype littermates, were subjected to HI at postnatal day 9. Brain injury at different ages was evaluated by the levels of cleaved caspase-3 and spectrin breakdown products at 24 hr; and histologically at 6 days or 3 months after HI. Multiple behavioral tests were performed at 3 months, prior to sacrifice. Loss of neuronal HIF-2α exacerbated brain injury during the acute (24 hr) and subacute phases (6 days), with a trend toward more severe volume loss in the adult brain. The long-term brain function for coordinated movement and recognition memory, however, were not impacted in the neuronal HIF-2α deficient mice. Our data suggest that, similar to HIF-1α, neuronal HIF-2α promotes cell survival in the immature mouse brain. The two HIF alpha isoforms may act through partially overlapping or distinct transcriptional targets to mediate their intrinsic protective responses against neonatal HI brain injury.

ObjectiveTo evaluate the association of therapeutic hypothermia with magnetic resonance imaging (MRI) findings and 30-month neurodevelopment in term neonatal encephalopathy.Study designCross-sectional analysis of 30-month neurodevelopment (IQR 19.0-31.4) in a prospective cohort of mild-to-severe neonatal encephalopathy imaged on day 4 (1993-2017 with institutional implementation of therapeutic hypothermia in 2007). MRI injury was classified as normal, watershed, or basal ganglia/thalamus. Abnormal motor outcome was defined as Bayley-II psychomotor developmental index

ImportanceCerebral palsy (CP) is the most common childhood physical disability. Early intervention for children younger than 2 years with or at risk of CP is critical. Now that an evidence-based guideline for early accurate diagnosis of CP exists, there is a need to summarize effective, CP-specific early intervention and conduct new trials that harness plasticity to improve function and increase participation. Our recommendations apply primarily to children at high risk of CP or with a diagnosis of CP, aged 0 to 2 years.ObjectiveTo systematically review the best available evidence about CP-specific early interventions across 9 domains promoting motor function, cognitive skills, communication, eating and drinking, vision, sleep, managing muscle tone, musculoskeletal health, and parental support.Evidence reviewThe literature was systematically searched for the best available evidence for intervention for children aged 0 to 2 years at high risk of or with CP. Databases included CINAHL, Cochrane, Embase, MEDLINE, PsycInfo, and Scopus. Systematic reviews and randomized clinical trials (RCTs) were appraised by A Measurement Tool to Assess Systematic Reviews (AMSTAR) or Cochrane Risk of Bias tools. Recommendations were formed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) framework and reported according to the Appraisal of Guidelines, Research, and Evaluation (AGREE) II instrument.FindingsSixteen systematic reviews and 27 RCTs met inclusion criteria. Quality varied. Three best-practice principles were supported for the 9 domains: (1) immediate referral for intervention after a diagnosis of high risk of CP, (2) building parental capacity for attachment, and (3) parental goal-setting at the commencement of intervention. Twenty-eight recommendations (24 for and 4 against) specific to the 9 domains are supported with key evidence: motor function (4 recommendations), cognitive skills (2), communication (7), eating and drinking (2), vision (4), sleep (7), tone (1), musculoskeletal health (2), and parent support (5).Conclusions and relevanceWhen a child meets the criteria of high risk of CP, intervention should start as soon as possible. Parents want an early diagnosis and treatment and support implementation as soon as possible. Early intervention builds on a critical developmental time for plasticity of developing systems. Referrals for intervention across the 9 domains should be specific as per recommendations in this guideline.

ObjectiveAlthough most seizures in neonates are due to acute brain injury, some represent the first sign of neonatal onset genetic epilepsies. Delay in recognition and lack of expert assessment of neonates with epilepsy may result in worse developmental outcomes. As in older children and adults, seizure semiology in neonates is an essential determinant in diagnosis. We aimed to establish whether seizure type at presentation in neonates can suggest a genetic etiology.MethodsWe retrospectively analyzed the clinical and electroencephalographic (EEG) characteristics of seizures in neonates admitted in two Level IV neonatal intensive care units, diagnosed with genetic epilepsy, for whom a video-EEG recording at presentation was available for review, and compared them on a 1:2 ratio with neonates with seizures due to stroke or hypoxic-ischemic encephalopathy.ResultsTwenty neonates with genetic epilepsy were identified and compared to 40 neonates with acute provoked seizures. Genetic epilepsies were associated with pathogenic variants in KCNQ2 (n = 12), KCNQ3 (n = 2), SCN2A (n = 2), KCNT1 (n = 1), PRRT2 (n = 1), and BRAT1 (n = 2). All neonates with genetic epilepsy had seizures with clinical correlates that were either tonic (18/20) or myoclonic (2/20). In contrast, 17 of 40 (42%) neonates with acute provoked seizures had electrographic only seizures, and the majority of the remainder had clonic seizures. Time to first seizure was longer in neonates with genetic epilepsies (median = 60 h of life) compared to neonates with acute provoked seizures (median = 15 h of life, p

Neonatal brain hypoxia-ischemia (HI) is a leading cause of morbidity and long-term disabilities in children. While we have made significant progress in describing HI mechanisms, the limited therapies currently offered for HI treatment in the clinical setting stress the importance of discovering new targetable pathways. Efferocytosis is an immunoregulatory and homeostatic process of clearance of apoptotic cells (AC) and cellular debris, best described in the brain during neurodevelopment. The therapeutic potential of stimulating defective efferocytosis has been recognized in neurodegenerative diseases. In this review, we will explore the involvement of efferocytosis after a stroke and HI as a promising target for new HI therapies.

BackgroundArginases (ARG isoforms, ARG-1/ARG-2) are key regulatory enzymes of inflammation and tissue repair; however, their role after neonatal brain hypoxia (H) and hypoxia-ischemia (HI) remains unknown.MethodsC57BL/6 mice subjected to the Vannucci procedure on postnatal day (P9) were sacrificed at different timepoints. The degree of brain damage was assessed histologically. ARG spatiotemporal localization was determined via immunohistochemistry. ARG expression was measured by Western blot and activity spectrophotometrically.ResultsARG isoform expression increased during neurodevelopment (P9-P17) in the cortex and hippocampus. This was suppressed with H and HI only in the hippocampus. In the cortex, both isoforms increased with H alone and only ARG-2 increased with HI at 3 days. ARG activity during neurodevelopment remained unchanged, but increased at 1 day with H and not HI. ARG-1 localized with microglia at the injury site as early as 4 h after injury, while ARG-2 localized with neurons.ConclusionsARG isoform expression increases with age from P9 to P17, but is suppressed by injury specifically in the hippocampus and not in the cortex. Both levels and activity of ARG isoforms increase with H, while ARG-1 immunolabelling is upregulated in the HI cortex. Evidently, ARG isoforms in the brain differ in spatiotemporal localization, expression, and activity during neurodevelopment and after injury.ImpactArginase isoforms change during neurodevelopment and after neonatal brain HI. This is the first study investigating the key enzymes of inflammation and tissue repair called arginases following murine neonatal brain HI. The highly region- and cell-specific expression suggests the possibility of specific functions of arginases. ARG-1 in microglia at the injury site may regulate neuroinflammation, while ARG-2 in neurons of developmental structures may impact neurodevelopment. While further studies are needed to describe the exact role of ARGs after neonatal brain HI, our study adds valuable data on anatomical localization and expression of ARGs in brain during development and after stroke.

The major pathway of brain cholesterol turnover relies on its hydroxylation into 24S-hydroxycholesterol (24S-HC) using brain-specific cytochrome P450 46A1 (CYP46A1). 24S-HC produced exclusively in the brain normally traverses the blood-brain barrier to enter the circulation to the liver for excretion; therefore, the serum 24S-HC level is an indication of cholesterol metabolism in the brain. We recently reported an upregulation of CYP46A1 following hypoxia-ischemia (HI) in the neonatal mouse brain and a correlation between serum 24S-HC levels and acute brain damage. Here, we performed a longitudinal study to investigate whether the serum 24S-HC concentrations predict long-term brain structural and functional outcomes. In postnatal day 9 mice subjected to HI, the serum 24S-HC levels increased at 6 h and 24 h after HI and correlated with the infarct volumes measured histologically or by T2-weighted MRI. The 24 h levels were associated with white matter volume loss quantified by MBP immunostaining and luxol fast blue staining. The animals with higher serum 24S-HC at 6 h and 24 h corresponded to those with more severe motor and cognitive deficits at 35-40 days after HI. These data suggest that 24S-HC could be a novel and early blood biomarker for severity of neonatal HI brain damage and associated functional impairments.

Background and purposePerinatal stroke is a common cause of life-long neurobehavioral compromise. Mesenchymal stromal cells (MSCs) and EPO (erythropoietin) have each demonstrated short-term benefit with delayed administration after stroke, and combination therapy may provide the most benefit. The purpose of this study is to determine the long-term histological and functional efficacy of enhanced, intranasal stem cell therapy (MSC preexposed to EPO) compared with standard MSC or multidose systemic EPO.MethodsTransient middle cerebral artery occlusion or sham surgery was performed in postnatal day (P) 10 Sprague-Dawley rats, who were treated with single-dose intranasal MSC, MSC preexposed to EPO (MSC/EPO), multidose systemic EPO (EPO3; 1000 u/kg per dose×3 every 72 hours), or cell-conditioned media on P13 (day 3 [P13-P19] for EPO), or on P17 (day 7 [P17-P23] for EPO). At 2 months of age, animals underwent novel object recognition, cylinder rearing, and open field testing to assess recognition memory, sensorimotor function, and anxiety in adulthood.ResultsMSC, MSC/EPO, and EPO3 improved brain volume when administered at 3 or 7 days after middle cerebral artery occlusion. MSC/EPO also enhanced long-term recognition memory with either day 3 or day 7 treatment, but EPO3 had the most long-term benefit, improving recognition memory and exploratory behavior and reducing anxiety.ConclusionsThese data suggest that single-dose MSC/EPO and multidose systemic EPO improve long-term neurobehavioral outcomes even when administration is delayed, although EPO was the most effective treatment overall. It is possible that EPO represents a final common pathway for improved long-term repair, although the specific mechanisms remain to be determined.

BackgroundHyperpolarized 13C spectroscopic magnetic resonance spectroscopy (MRS) is an advanced imaging tool that may provide important real-time information about brain metabolism.MethodsMice underwent unilateral hypoxia-ischemia (HI) on postnatal day (P)10. Injured and sham mice were scanned at P10, P17, and P31. We used hyperpolarized 13C MRS to investigate the metabolic exchange of pyruvate to lactate in real time during brain development following HI. 13C-1-labeled pyruvate was hyperpolarized and injected into the tail vein through a tail-vein catheter. Chemical-shift imaging was performed to acquire spectral-spatial information of the metabolites in the brain. A voxel placed on each of the injured and contralateral hemispheres was chosen for comparison. The difference in pyruvate delivery and lactate to pyruvate ratio was calculated for each of the voxels at each time point. The normalized lactate level of the injured hemisphere was also calculated for each mouse at each of the scanning time points.ResultsThere was a significant reduction in pyruvate delivery and a higher lactate to pyruvate ratio in the ipsilateral (HI) hemisphere at P10. The differences decreased at P17 and disappeared at P31. The normalized lactate level in the injured hemisphere increased from P10 to P31 in both sham and HI mice without brain injury.ConclusionWe describe a method for detecting and monitoring the evolution of HI injury during brain maturation which could prove to be an excellent biomarker of injury.

BackgroundSixty percent of infants with severe neonatal hypoxic-ischemic encephalopathy die, while most survivors have permanent disabilities. Treatment for neonatal hypoxic-ischemic encephalopathy is limited to therapeutic hypothermia, but it does not offer complete protection. Here, we investigated whether hypoxia-inducible factor (HIF) promotes cell survival and suggested neuroprotective strategies.PurposeHIF-1α-deficient mice have increased brain injury after neonatal hypoxia-ischemia (HI), and the role of HIF-2α in HI is not well characterized. Copper-zinc superoxide dismutase (SOD)1 overexpression is not beneficial in neonatal HI. The expression of HIF-1α and HIF-2α was measured in SOD1 overexpressing mice and compared to wild-type littermates to see if alteration in expression explains this lack of benefit.MethodsOn postnatal day 9, C57Bl/6 mice were subjected to HI, and protein expression was measured by western blotting in the ipsilateral cortex of wild-type and SOD1 overexpressing mice to quantify HIF-1α and HIF-2α. Spectrin expression was also measured to characterize the mechanism of cell death.ResultsHIF-1α protein expression did not significantly change after HI injury in the SOD1-overexpressing or wild-type mouse cortex. However, HIF-2α protein expression increased 30 minutes after HI injury in the wild-type and SOD1-overexpressing mouse cortex and decreased to baseline value at 24 hours after HI injury. Spectrin 145/150 expression did not significantly change after HI injury in the SOD1- overexpressing or wild-type mouse cortex. However, spectrin 120 expression increased in both wild-type and SOD1-overexpressing mouse at 4 hours after HI, which decreased by 24 hours, indicating a greater role of apoptotic cell death.ConclusionHIF-1α and HIF-2α may promote cell survival in neonatal HI in a cell-specific and regional fashion. Our findings suggest that early HIF-2α upregulation precedes apoptotic cell death and limits necrotic cell death. However, the influence of SOD was not clarified; it remains an intriguing factor in neonatal HI.

BackgroundNeonatal seizures are associated with adverse neurologic sequelae including epilepsy in childhood. Here we aim to determine whether levels of cytokines in neonates with brain injury are associated with acute symptomatic seizures or remote epilepsy.MethodsThis is a cohort study of term newborns with encephalopathy at UCSF between 10/1993 and 1/2000 who had dried blood spots. Maternal, perinatal/postnatal, neuroimaging, and epilepsy variables were abstracted by chart review. Logistic regression was used to compare levels of cytokines with acute seizures and the development of epilepsy.ResultsIn a cohort of 26 newborns with neonatal encephalopathy at risk for hypoxic ischemic encephalopathy with blood spots for analysis, diffuse alterations in both pro- and anti-inflammatory cytokine levels were observed between those with (11/28, 39%) and without acute symptomatic seizures. Seventeen of the 26 (63%) patients had >2 years of follow-up and 4/17 (24%) developed epilepsy. Higher levels of pro-inflammatory cytokines IL-6 and TNF-α within the IL-1β pathway were significantly associated with epilepsy.ConclusionsElevations in pro-inflammatory cytokines in the IL-1β pathway were associated with later onset of epilepsy. Larger cohort studies are needed to confirm the predictive value of these circulating biomarkers.

Neonatal brain injury affects thousands of babies each year and may lead to long-term and permanent physical and neurological problems. Currently, therapeutic hypothermia is standard clinical care for term newborns with moderate to severe neonatal encephalopathy. Nevertheless, it is not completely protective, and additional strategies to restore and promote regeneration are urgently needed. One way to ensure recovery following injury to the immature brain is to augment endogenous regenerative pathways. However, novel strategies such as stem cell therapy, gene therapies and nanotechnology have not been adequately explored in this unique age group. In this perspective review, we describe current efforts that promote neuroprotection and potential targets that are unique to the developing brain, which can be leveraged to facilitate neuroregeneration.

Hypoxia-inducible factor 1α (HIF1α) is a key regulator of oxygen homeostasis, and its target genes mediate adaptive, protective, and pathological processes. The role of HIF1α in neuronal survival is controversial and the brain maturation stage is important in determining its function in brain ischemia or hypoxia-ischemia (HI). In this study, we used neuron-specific HIF1α knockout mice at postnatal day 9 (P9), and immature cortical neurons (days 7-8 in vitro) treated with the HIF1α inhibitor 2-methoxyestradiol (2ME2) or stabilizer dimethyloxalylglycine (DMOG), to examine the function of neuronal HIF1α in neonatal HI in vivo (Vannucci model) and in vitro (oxygen glucose deprivation, OGD). Inhibition of HIF1α with 2ME2 in primary neurons or deletion of neuronal HIF1α in P9 mice increased both necrotic and apoptotic cell death following HI, as evaluated by the protein levels of 145/150-kDa and 120-kDa spectrin breakdown products 24 h after HI. DMOG attenuated neuronal death right after OGD. Acute pharmacological manipulation of HIF1α synchronously regulated the expression of its targets, vascular endothelial growth factor (VEGF) and erythropoietin (Epo), in the same manner. The in vivo findings agree with our previous data using the same HIF1α-deficient mice at an earlier age. This study confirms the role of neuronal HIF1α signaling in the endogenous protective responses following HI in the developing brain.

Purpose- Much has transpired since the last scientific statement on pediatric stroke was published 10 years ago. Although stroke has long been recognized as an adult health problem causing substantial morbidity and mortality, it is also an important cause of acquired brain injury in young patients, occurring most commonly in the neonate and throughout childhood. This scientific statement represents a synthesis of data and a consensus of the leading experts in childhood cardiovascular disease and stroke. Methods- Members of the writing group were appointed by the American Heart Association Stroke Council's Scientific Statement Oversight Committee and the American Heart Association's Manuscript Oversight Committee and were chosen to reflect the expertise of the subject matter. The writers used systematic literature reviews, references to published clinical and epidemiology studies, morbidity and mortality reports, clinical and public health guidelines, authoritative statements, personal files, and expert opinion to summarize existing evidence and to indicate gaps in current knowledge. This scientific statement is based on expert consensus considerations for clinical practice. Results- Annualized pediatric stroke incidence rates, including both neonatal and later childhood stroke and both ischemic and hemorrhagic stroke, range from 3 to 25 per 100 000 children in developed countries. Newborns have the highest risk ratio: 1 in 4000 live births. Stroke is a clinical syndrome. Delays in diagnosis are common in both perinatal and childhood stroke but for different reasons. To develop new strategies for prevention and treatment, disease processes and risk factors that lead to pediatric stroke are discussed here to aid the clinician in rapid diagnosis and treatment. The many important differences that affect the pathophysiology and treatment of childhood stroke are discussed in each section. Conclusions- Here we provide updates on perinatal and childhood stroke with a focus on the subtypes, including arterial ischemic, venous thrombotic, and hemorrhagic stroke, and updates in regard to areas of childhood stroke that have not received close attention such as sickle cell disease. Each section is highlighted with considerations for clinical practice, attendant controversies, and knowledge gaps. This statement provides the practicing provider with much-needed updated information in this field.

BackgroundTo assess whether postnatal plasma cholesterol levels are associated with microstructural and macrostructural regional brain development in preterm newborns.MethodsSixty preterm newborns (born 24-32 weeks gestational age) were assessed using MRI studies soon after birth and again at term-equivalent age. Blood samples were obtained within 7 days of each MRI scan to analyze for plasma cholesterol and lathosterol (a marker of endogenous cholesterol synthesis) levels. Outcomes were assessed at 3 years using the Bayley Scales of Infant Development, Third Edition.ResultsEarly plasma lathosterol levels were associated with increased axial and radial diffusivities and increased volume of the subcortical white matter. Early plasma cholesterol levels were associated with increased volume of the cerebellum. Early plasma lathosterol levels were associated with a 2-point decrease in motor scores at 3 years.ConclusionsHigher early endogenous cholesterol synthesis is associated with worse microstructural measures and larger volumes in the subcortical white matter that may signify regional edema and worse motor outcomes. Higher early cholesterol is associated with improved cerebellar volumes. Further work is needed to better understand how the balance of cholesterol supply and endogenous synthesis impacts preterm brain development, especially if these may be modifiable factors to improve outcomes.

Acute chorioamnionitis refers to the neutrophilic inflammation of the placental tissues thought to result from an ascending bacterial infection. It is considered a major factor associated with pretermbirth and has been estimated to occur in 40%to 80% of preterm deliveries. Chorioamnionitis is associated with several adverse neonatal outcomes, including respiratory distress syndrome, sepsis, bronchopulmonary dysplasia, and death. Clinical studies examining brain injury and neurodevelopmental outcomes among infants with chorioamnionitis have yielded inconsistent results. Most of these studies have focused on intraventricular hemorrhage (IVH) and cystic periventricular leukomalacia. Because the incidence of cystic periventricular leukomalacia has greatly decreased during the past decades concurrent with improvements in neonatal intensive care, punctate white matter injury (WMI) is increasingly recognized as the most prevalent pattern of brain injury among preterm neonates. The researchers performed a prospective cohort study conducted across 3 academic centers in Canada, the Netherlands, and the United States. Children who were born preterm (24-32 weeks' gestation) and who had undergone a placental pathologic evaluation, magnetic resonance imaging (MRI) as soon as clinically stable, and Bayley Scales of Infant and Toddler Development, 3rd Edition (Bayley III) assessments between 18 and 24 months' corrected age (CA) were included. Magnetic resonance imaging scans were assessed for grade of IVH and volume of punctate WMI. Data analysis occurred between December 2016 and January 2018. Final multivariable analyses examining the association of chorioamnionitis with motor and cognitive outcomes accounted for academic center and perinatal and postnatal factors. PunctateWMI volume and IVH detected on neonatalMRI scans were used tomeasure the results,withmotor and cognitive outcomes defined using Bayley III assessments conducted among these children between 18 and 24 months' CA. There were 448 preterm infants (24-32 weeks' gestation) in the total cohort. Infants were included in the analysis if they had undergone placental pathologic assessments, early brainMRI, and 18 to 24 months of follow-up.Among the cohorts fromthe 3 academic centers, infants with evidence of a congenital infection, genetic syndrome, or large parenchymal hemorrhagic infarction (>2 cm) were excluded. Each placenta was sent fresh for macroscopic and microscopic analyses, which were conducted using the same clinical protocols at each center. Histologic chorioamnionitis was defined by clinical pathologists using strict criteria and the degree of placental inflammation was scored.

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.

Brain damage after hypoxia-ischemia (HI) occurs in an age-dependent manner. Neuroprotective strategies assumed to be effective in adults might have deleterious effects in the immature brain. In order to create effective therapies, the complex pathophysiology of HI in the developing brain requires exploring new mechanisms. Critical determinants of neuronal survival after HI are the extent of vascular dysfunction, inflammation, and oxidative stress, followed later by tissue repair. The key enzyme of these processes in the human body is arginase (ARG) that acts via the bioavailability of nitric oxide, and the synthesis of polyamines and proline. ARG is expressed throughout the brain in different cells. However, little is known about the effect of ARG in pathophysiological states of the brain, especially hypoxia-ischemia. Here, we summarize the role of ARG during neurodevelopment as well as in various brain pathologies.

The N-methyl-d-aspartate-type glutamate receptor (NMDAR)-associated multiprotein complexes are indispensable for synaptic plasticity and cognitive functions. While purification and proteomic analyses of these signaling complexes have been performed in adult rodent and human brain, much less is known about the protein composition of NMDAR complexes in the developing brain and their modifications by neonatal hypoxic-ischemic (HI) brain injury. In this study, the postsynaptic density proteins were prepared from postnatal day 9 naïve, sham-operated and HI-injured mouse cortex. The GluN2B-containing NMDAR complexes were purified by immunoprecipitation with a mouse GluN2B antibody and subjected to mass spectrometry analysis for determination of the GluN2B binding partners. A total of 71 proteins of different functional categories were identified from the naïve animals as native GluN2B-interacting partners in the developing mouse brain. Neonatal HI reshaped the postsynaptic GluN2B interactome by recruiting new proteins, including multiple kinases, into the complexes; and modifying the existing associations within 1h of reperfusion. The early responses of postsynaptic NMDAR complexes and their related signaling networks may contribute to molecular processes leading to cell survival or death, brain damage and/or neurological disorders in term infants with neonatal encephalopathy.

Brain development is an energy-expensive process. Although glucose is irreplaceable, the developing brain utilizes a variety of substrates such as lactate and the ketone bodies, β-hydroxybutyrate and acetoacetate, to produce energy and synthesize the structural components necessary for cerebral maturation. When oxygen and nutrient supplies to the brain are restricted, as in neonatal hypoxia-ischemia (HI), cerebral energy metabolism undergoes alterations in substrate use to preserve the production of adenosine triphosphate. These changes have been studied by in situ biochemical methods that yielded valuable quantitative information about high-energy and glycolytic metabolites and established a temporal profile of the cerebral metabolic response to hypoxia and HI. However, these analyses relied on terminal experiments and averaging values from several animals at each time point as well as challenging requirements for accurate tissue processing.More recent methodologies have focused on in vivo longitudinal analyses in individual animals. The emerging field of metabolomics provides a new investigative tool for studying cerebral metabolism. Magnetic resonance spectroscopy (MRS) has enabled the acquisition of a snapshot of the metabolic status of the brain as quantifiable spectra of various intracellular metabolites. Proton (1H) MRS has been used extensively as an experimental and diagnostic tool of HI in the pursuit of markers of long-term neurodevelopmental outcomes. Still, the interpretation of the metabolite spectra acquired with 1H MRS has proven challenging, due to discrepancies among studies, regarding calculations and timing of measurements. As a result, the predictive utility of such studies is not clear. 13C MRS is methodologically more challenging, but it provides a unique window on living tissue metabolism via measurements of the incorporation of 13C label from substrates into brain metabolites and the localized determination of various metabolic fluxes. The newly developed hyperpolarized 13C MRS is an exciting method for assessing cerebral metabolism in vivo, that bears the advantages of conventional 13C MRS but with a huge gain in signal intensity and much shorter acquisition times. The first part of this review article provides a brief description of the findings of biochemical and imaging methods over the years as well as a discussion of their associated strengths and pitfalls. The second part summarizes the current knowledge on cerebral metabolism during development and HI brain injury.

BackgroundHuman studies investigating the link between postnatal polyunsaturated fatty acids and preterm brain growth are limited, despite emerging evidence of potential effects on outcomes.MethodsSixty preterm neonates

Neonatal hypoxic-ischemic brain injury is commonly studied by means of the Vannucci procedure in mice or rats (unilateral common carotid artery occlusion followed by hypoxia). Previously, we modified the postnatal day 7 (P7) rat procedure for use in mice, and later demonstrated that genetic strain strongly influences the degree of brain injury in the P7 mouse model of hypoxia-ischemia (HI). Recently, the P9 or P10 mouse brain was recognized as the developmental equivalent of a term neonatal human brain, rather than P7. Consequently, the Vannucci procedure has again been modified, and a commonly used protocol employs 10% oxygen for 50 min in C57Bl/6 mice. Strain differences have yet to be described for the P9/P10 mouse model. In order to determine if the strain differences we previously reported in the P7 mouse model are present in the P9 model, we compared 2 commonly used strains, CD1 and C57Bl/6J, in both the P7 (carotid ligation [in this case, right] followed by exposure to 8% oxygen for 30 min) and P9 (carotid ligation [in this case left] followed by exposure to 10% oxygen) models of HI. Experiments using the P7 model were performed in 2001-2012 and those using the P9 model were performed in 2012-2016. Five to seven days after the HI procedure, mice were perfused with 4% paraformaldehyde, their brains were sectioned on a Vibratome (50 µm) and alternate sections were stained with Perl's iron stain or cresyl violet. Brain sections were examined microscopically and scored for the degree of injury. Since brains in the P7 group had been scored previously with a slightly different system, they were reanalyzed using our current scoring system which scores injury in 11 regions: the anterior, middle, and posterior cortex; the anterior, middle, and posterior striatum; CA1, CA2, CA3, and the dentate gyrus of the hippocampus and thalamus, on a scale from 0 (none) to 3 (cystic infarct) for a total score of 0-33. Brains in the P9 group were scored with the same system. Given the same insult, the P7 CD1 mice had greater injury than the C57Bl/6J mice, which agrees with our previous findings. The P9 CD1 mice also had greater injury than the C57Bl/6J mice. This study confirms that CD1 mice are more susceptible to injury than C57Bl/6J mice and that strain selection is important when using mouse models of HI.

Hypoxia-inducible factor 1α (HIF1α) is a key regulator of oxygen homeostasis, and its target genes mediate adaptive, protective, and pathological processes. The role of HIF1α in neuronal survival is controversial and the brain maturation stage is important in determining its function in brain ischemia or hypoxia-ischemia (HI). In this study, we used neuron-specific HIF1α knockout mice at postnatal day 9 (P9), and immature cortical neurons (days 7-8 in vitro) treated with the HIF1α inhibitor 2-methoxyestradiol (2ME2) or stabilizer dimethyloxalylglycine (DMOG), to examine the function of neuronal HIF1α in neonatal HI in vivo (Vannucci model) and in vitro (oxygen glucose deprivation, OGD). Inhibition of HIF1α with 2ME2 in primary neurons or deletion of neuronal HIF1α in P9 mice increased both necrotic and apoptotic cell death following HI, as evaluated by the protein levels of 145/150-kDa and 120-kDa spectrin breakdown products 24 h after HI. DMOG attenuated neuronal death right after OGD. Acute pharmacological manipulation of HIF1α synchronously regulated the expression of its targets, vascular endothelial growth factor (VEGF) and erythropoietin (Epo), in the same manner. The in vivo findings agree with our previous data using the same HIF1α-deficient mice at an earlier age. This study confirms the role of neuronal HIF1α signaling in the endogenous protective responses following HI in the developing brain.

BackgroundTherapeutic hypothermia (TH) is the standard of care for neonates with hypoxic-ischemic encephalopathy, but it is not fully protective in the clinical setting. Hypoxia-ischemia (HI) may cause white matter injury (WMI), leading to neurological and cognitive dysfunction.MethodsP9 mice were subjected to HI as previously described. Pups underwent 3.5 h of systemic hypothermia or normothermia. Cresyl violet and Perl's iron staining for histopathological scoring of brain sections was completed blindly on all brains. Immunocytochemical (ICC) staining for myelin basic protein (MBP), microglia (Iba1), and astrocytes (glia fibrillary acidic protein (GFAP)) was performed on adjacent sections. Volumetric measurements of MBP coverage were used for quantitative analysis of white matter.ResultsTH provided neuroprotection by injury scoring for the entire group (n=44; P

ImportanceCerebral palsy describes the most common physical disability in childhood and occurs in 1 in 500 live births. Historically, the diagnosis has been made between age 12 and 24 months but now can be made before 6 months' corrected age.ObjectivesTo systematically review best available evidence for early, accurate diagnosis of cerebral palsy and to summarize best available evidence about cerebral palsy-specific early intervention that should follow early diagnosis to optimize neuroplasticity and function.Evidence reviewThis study systematically searched the literature about early diagnosis of cerebral palsy in MEDLINE (1956-2016), EMBASE (1980-2016), CINAHL (1983-2016), and the Cochrane Library (1988-2016) and by hand searching. Search terms included cerebral palsy, diagnosis, detection, prediction, identification, predictive validity, accuracy, sensitivity, and specificity. The study included systematic reviews with or without meta-analyses, criteria of diagnostic accuracy, and evidence-based clinical guidelines. Findings are reported according to the PRISMA statement, and recommendations are reported according to the Appraisal of Guidelines, Research and Evaluation (AGREE) II instrument.FindingsSix systematic reviews and 2 evidence-based clinical guidelines met inclusion criteria. All included articles had high methodological Quality Assessment of Diagnostic Accuracy Studies (QUADAS) ratings. In infants, clinical signs and symptoms of cerebral palsy emerge and evolve before age 2 years; therefore, a combination of standardized tools should be used to predict risk in conjunction with clinical history. Before 5 months' corrected age, the most predictive tools for detecting risk are term-age magnetic resonance imaging (86%-89% sensitivity), the Prechtl Qualitative Assessment of General Movements (98% sensitivity), and the Hammersmith Infant Neurological Examination (90% sensitivity). After 5 months' corrected age, the most predictive tools for detecting risk are magnetic resonance imaging (86%-89% sensitivity) (where safe and feasible), the Hammersmith Infant Neurological Examination (90% sensitivity), and the Developmental Assessment of Young Children (83% C index). Topography and severity of cerebral palsy are more difficult to ascertain in infancy, and magnetic resonance imaging and the Hammersmith Infant Neurological Examination may be helpful in assisting clinical decisions. In high-income countries, 2 in 3 individuals with cerebral palsy will walk, 3 in 4 will talk, and 1 in 2 will have normal intelligence.Conclusions and relevanceEarly diagnosis begins with a medical history and involves using neuroimaging, standardized neurological, and standardized motor assessments that indicate congruent abnormal findings indicative of cerebral palsy. Clinicians should understand the importance of prompt referral to diagnostic-specific early intervention to optimize infant motor and cognitive plasticity, prevent secondary complications, and enhance caregiver well-being.

Cell therapy has emerged as a potential treatment for many neurodegenerative diseases including stroke and neonatal ischemic brain injury. Delayed intranasal administration of mesenchymal stem cells (MSCs) after experimental hypoxia-ischemia and after a transient middle cerebral artery occlusion (tMCAO) in neonatal rats has shown improvement in long-term functional outcomes, but the effects of MSCs on white matter injury (WMI) are insufficiently understood. In this study we used longitudinal T2-weighted (T2W) and diffusion tensor magnetic resonance imaging (MRI) to characterize chronic injury after tMCAO induced in postnatal day 10 (P10) rats and examined the effects of delayed MSC administration on WMI, axonal coverage, and long-term somatosensory function. We show unilateral injury- and region-dependent changes in diffusion fraction anisotropy 1 and 2 weeks after tMCAO that correspond to accumulation of degraded myelin basic protein, astrocytosis, and decreased axonal coverage. With the use of stringent T2W-based injury criteria at 72 hr after tMCAO to randomize neonatal rats to receive intranasal MSCs or vehicle, we show that a single MSC administration attenuates WMI and enhances somatosensory function 28 days after stroke. A positive correlation was found between MSC-enhanced white matter integrity and functional performance in injured neonatal rats. Collectively, these data indicate that the damage induced by tMCAO progresses over time and is halted by administration of MSCs. © 2016 Wiley Periodicals, Inc.

Hypoxic‐ischemic encephalopathy (HIE) is a common neurological syndrome in newborns with high mortality and morbidity. Therapeutic hypothermia (TH), which is standard of care for HIE, mitigates brain injury by suppressing anaerobic metabolism. However, more than 40% of HIE neonates have a poor outcome, even after TH. This study aims to provide metabolic biomarkers for predicting the outcomes of hypoxia‐ischemia (HI) after TH using hyperpolarized [1‐13C] pyruvate magnetic resonance spectroscopy. Postnatal day 10 (P10) mice with HI underwent TH at 1 h and were scanned at 6–8 h (P10), 24 h (P11), 7 days (P17), and 21 days (P31) post‐HI on a 14.1‐T NMR spectrometer. The metabolic images were collected, and the conversion rate from pyruvate to lactate and the ratio of lactate to pyruvate in the injured left hemisphere (kPL(L) and Lac/Pyr(L), respectively) were calculated at each timepoint. The outcomes of TH were determined by the assessments of brain injury on T2‐weighted images and behavioral tests at later timepoint. kPL(L) and Lac/Pyr(L) over time between the good‐outcome and poor‐outcome groups and across timepoints within groups were analyzed. We found significant differences in temporal trends of kPL(L) and Lac/Pyr(L) between groups. In the good‐outcome group, kPL(L) increased until P31 with a significantly higher value at P31 compared with that at P10, while the level of Lac/Pyr(L) at P31 was notably higher than those at all other timepoints. In the poor‐outcome group, kPL(L) and Lac/Pyr(L) increased within 24 h. The kPL(L) value at P11 was considerably higher compared with P10. Discrete temporal changes of kPL(L) and Lac/Pyr(L) after TH between the good‐outcome and poor‐outcome groups were seen as early as 24 h after HI, reflecting various TH effects on brain anaerobic metabolism, which may provide insights for early screening for response to TH. [ABSTRACT FROM AUTHOR]

Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding of protein composition and signal networks in the adult brain, which underlie neuronal plasticity and neurodegenerative or psychiatric disorders. However, there is a paucity of knowledge about the architecture and organization of PSDs in the immature brain, and how it is modified by brain injury in an early developing stage. Mass spectrometry (MS)-based proteomic analysis was performed on PSDs prepared from cortices of postnatal day 9 naïve mice or pups which had suffered hypoxic-ischemic (HI) brain injury. 512 proteins of different functional groups were identified from PSDs collected 1 h after HI injury, among which 60 have not been reported previously. Seven newly identified proteins involved in neural development were highlighted. HI injury increased the yield of PSDs at early time points upon reperfusion, and multiple proteins were recruited into PSDs following the insult. Quantitative analysis was performed using spectral counting, and proteins whose relative expression was more than 50% up- or downregulated compared to the sham animals 1 h after HI insult were reported. Validation with Western blotting demonstrated changes in expression and phosphorylation of the N-methyl-D-aspartate receptor, activation of a series of postsynaptic protein kinases and dysregulation of scaffold and adaptor proteins in response to neonatal HI insult. This work, along with other recent studies of synaptic protein profiling in the immature brain, builds a foundation for future investigation on the molecular mechanisms underlying developing plasticity. Furthermore, it provides insights into the biochemical changes of PSDs following early brain hypoxia-ischemia, which is helpful for understanding not only the injury mechanisms, but also the process of repair or replenishment of neuronal circuits during recovery from brain damage.

The neonatal brain is highly susceptible to oxidative stress as developing endogenous antioxidant mechanisms are overwhelmed. In the neonate, superoxide dismutase (SOD) overexpression worsens hypoxic-ischemic injury due to H2O2 accumulation in the brain. Erythropoietin (EPO) is upregulated in 2 phases after HI, early (4 h) and late (7 days), and exogenous EPO has been effective in reducing the injury, possibly through reducing oxidative stress. We hypothesized that exogenous EPO would limit injury from excess H2O2 seen in SOD1-overexpressing mice, and thus enhance recovery after HI. We first wanted to confirm our previous findings in postnatal day 7 (P7) SOD-tg (CD1) mice using a P9 model of the Vannucci procedure of HI with SOD-tg mice from a different background strain (C57Bl/6), and then determine the efficacy of EPO treatment in this strain and their wild-type (WT) littermates. Thus, mice overexpressing copper/zinc SOD1 were subjected to HI, modified for the P9 mouse, and recombinant EPO (5 U/g) or vehicle (saline) was administered intraperitoneally 3 times: at 0 h, 24 h, and 5 days. Injury was assessed 7 days after HI. In addition, protein expression for EPO and EPO receptor was assessed in the cortex and hippocampus 24 h after HI. With the moderate insult, the SOD-tg mice had greater injury than the WT overall, confirming our previous results, as did the hippocampus and striatum when analyzed separately, but not the cortex or thalamus. EPO treatment worsened injury in SOD-tg overall and in the WT and SOD-tg hippocampus and striatum. With the more severe insult, all groups had greater injury than with the moderate insult, but differences between SOD-tg and WT were no longer observed and EPO treatment had no effect. Increased protein expression of EPO was observed in the cortex of SOD-tg mice given recombinant human EPO compared to SOD-tg given vehicle. This study confirms our previous results showing greater injury with SOD overexpression in the neonatal brain after HI at P7 in a different strain. These results also suggest that EPO treatment cannot ameliorate the damage seen in situations where there is excess H2O2 accumulation, and it may exacerbate injury in settings of extreme oxidative stress.

The neonatal brain is especially susceptible to oxidative stress owing to its reduced antioxidant capacity. Following hypoxic-ischemic (HI) injury, for example, there is a prolonged elevation in levels of hydrogen peroxide (H2O2) in the immature brain compared to the adult brain, resulting in lasting injury that can lead to life-long disability or morbidity. Erythropoietin (Epo) is one of few multifaceted treatment options that have been promising enough to trial in the clinic for both term and preterm brain injury. Despite this, there is a lack of clear understanding of how Epo modulates glial cell activity following oxidative injury, specifically, whether it affects microglia (Mg) and astrocytes (Ast) differently. Using an in vitro approach using primary murine Mg and Ast subjected to H2O2 injury, we studied the oxidative and inflammatory responses of Mg and Ast to recombinant murine (rm)Epo treatment. We found that Epo protects Ast from H2O2 injury (p < 0.05) and increases secreted nitric oxide levels in these cells (p < 0.05) while suppressing intracellular reactive oxygen species (p < 0.05) and superoxide ion (p < 0.05) levels only in Mg. Using a multiplex analysis, we noted that although H2O2 induced the levels of several chemokines, rmEpo did not have any significant specific effects on their levels, either with or without the presence of conditioned medium from injured neurons (NCM). Ultimately, it appears that rmEpo has pleiotropic effects based on the cell type; it has a protective effect on Ast but an antioxidative effect only on Mg without any significant modulation of chemokine and cytokine levels in either cell type. These findings highlight the importance of considering all cell types when assessing the benefits and pitfalls of Epo use.