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Organisms have homeostatic mechanisms to respond to cold to ensure survival including the activation of the neuroprotective mild hypothermia response (MHR) in mammals at 32C. We show activation of the MHR by one FDA-approved medication, Entacapone, a proof-of-principle that the MHR can be medically manipulated. Utilizing a forward CRISPR-Cas9 mutagenesis screen, we identify the histone lysine methyltransferase SMYD5 as an epigenetic gatekeeper of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia but not at 32C. This repression is mirrored by temperature-dependent levels of H3K36me3 at the SP1-locus and globally indicating the mammalian MHR is regulated at the level of histone modifications. We identified 45 additional SMYD5-temperature dependent genes suggesting a broader MHR-related role for SMYD5. Our study provides an example of how the epigenetic machinery integrates environmental cues into the genetic circuitry of mammalian cells and suggests novel therapeutic avenues for neuroprotection after catastrophic events.

Identifying the hemodynamic range that best supports cerebral perfusion using near infrared spectroscopy (NIRS) autoregulation monitoring is a potential physiologic marker for neonatal hypoxic-ischemic encephalopathy (HIE) during therapeutic hypothermia. However, an optimal autoregulation monitoring algorithm has not been identified for neonatal clinical medicine. We tested whether the hemoglobin volume phase (HVP), hemoglobin volume (HVx), and pressure passivity index (PPI) identify changes in autoregulation that are associated with brain injury on MRI or death. The HVP measures the phase difference between a NIRS metric of cerebral blood volume, the total hemoglobin (THb), and mean arterial blood pressure (MAP) at the frequency of maximum coherence. The HVx is the correlation coefficient between MAP and THb. The PPI is the percentage of coherent MAP-DHb (difference between oxygenated and deoxygenated hemoglobin, a marker of cerebral blood flow) epochs in a chosen time period. Neonates cooled for HIE were prospectively enrolled in an observational study in two neonatal intensive care units. In analyses adjusted for study site and encephalopathy level, all indices detected relationships between poor autoregulation in the first 6 h after rewarming with a higher injury score on MRI. Only HVx and PPI during hypothermia and the PPI during rewarming identified autoregulatory dysfunction associated with a poor outcome independent of study site and encephalopathy level. Our findings suggest that the accuracy of mathematical autoregulation algorithms in detecting the risk of brain injury or death may depend on temperature and postnatal age. Extending autoregulation monitoring beyond the standard 72 h of therapeutic hypothermia may serve as a method to provide personalized care by assessing the need for and efficacy of future therapies after the hypothermia treatment phase.

Neonatal hypoxic-ischemic encephalopathy (HIE) causes lifelong neurologic disability. Despite the use of therapeutic hypothermia, memory deficits and executive functions remain severely affected. Cholinergic neurotransmission from the basal forebrain to neocortex and hippocampus is central to higher cortical functions. We examined the basal forebrain by light microscopy and reported loss of choline acetyltransferase-positive (ChAT)+ neurons, at postnatal day (P) 40, in the ipsilateral medial septal nucleus (MSN) after neonatal hypoxia-ischemia (HI) in mice. There was no loss of ChAT+ neurons in the ipsilateral nucleus basalis of Meynert (nbM) and striatum. Ipsilateral striatal and nbM ChAT+ neurons were abnormal with altered immunoreactivity for ChAT, shrunken and crenated somas, and dysmorphic appearing dendrites. Using confocal images with 3D reconstruction, nbM ChAT+ dendrites in HI mice were shorter than sham (p = .0001). Loss of ChAT+ neurons in the MSN directly correlated with loss of ipsilateral hippocampal area. In the nbM and striatum, percentage of abnormal ChAT+ neurons correlated with loss of ipsilateral cerebral cortical and striatal area, respectively. Acetylcholinesterase (AChE) activity increased in adjacent ipsilateral cerebral cortex and hippocampus and the increase was linearly related to loss of cortical and hippocampal area. Numbers and size of cathepsin D+ lysosomes increased in large neurons in the ipsilateral nbM. After neonatal HI, abnormalities were found throughout the major cholinergic systems in relationship to amount of forebrain area loss. There was also an upregulation of cathepsin D+ particles within the nbM. Cholinergic neuropathology may underlie the permanent dysfunction in learning, memory, and executive function after neonatal brain injury.

Background To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. Methods Plasma samples from the NE (n = 45, day of life 0-1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). Results Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. Conclusions Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors. Impact Plasma metabolites may help to predict neurological outcomes in NE and supplement current clinical predictors. Current metabolomics research is limited in terms of clinical application and association with long-term outcomes. Our study presents novel associations of plasma metabolites from the first 24 h of life and 2-year neurodevelopmental outcomes for infants with NE. Our metabolomics discovery provides insight into possible disease mechanisms and methods to rescue and/or supplement metabolic pathways involved in NE. Our metabolomics discovery of metabolic pathway supplementations and/or rescue mechanisms may serve as adjunctive therapies for NE.

To determine a safe dose of clonidine (CLON) to be used in infants with hypoxic ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). A pilot prospective study was performed to determine the effect of CLON on autonomic parameters, the pharmacokinetics (PK) of CLON, and the amount of morphine (MOR) given “as needed” for shivering and agitation in a cohort of infants (n = 12) with HIE undergoing TH compared to a historical control group (n = 28). The CLON group received less “as needed” MOR than the MOR-only group for agitation/shivering (p

OBJECTIVE: To determine if blood biomarkers measured at delivery and shortly after birth can identify growth restricted infants at risk for developing severe brain injury. STUDY DESIGN: In a cohort of very low birth weight neonates, fetal growth restricted (FGR) (birth weight < 10%) were compared to non-FGR neonates, and within the FGR group those with brain injury were compared to those without. Biomarkers were measured in cord blood at delivery, and daily for the 1st 5 days of life. RESULT: FGR was associated with significantly higher levels of interleukin (IL)-6, IL-8, IL-10 and lower levels of vascular endothelial growth factor (VEGF). FGR and brain injury were associated with significantly higher levels of IL-6, IL-8, IL-10 and glial fibrillary acidic protein (GFAP). CONCLUSION: Interleukins may be involved in a common pathway contributing to both the development of growth restriction and brain injury, and GFAP may help identify brain injury within this growth restricted group., PRECIS: Biomarkers measured in cord blood at delivery and neonatal serum after birth may identify growth restricted infants at risk for developing severe brain injury.

Background: Cooling delays, temperature outside 33–34°C, and blood pressure below the mean arterial blood pressure with optimal cerebral autoregulation (MAPOPT) might diminish neuroprotection from therapeutic hypothermia in neonates with hypoxic-ischemic encephalopathy (HIE). We hypothesized that longer time to reach temperature

Cerebral palsy (CP) is the most common cause of physical disability for children worldwide. Many infants and toddlers are not diagnosed with CP until they fail to achieve obvious motor milestones. Currently, there are no effective pharmacologic interventions available for infants and toddlers to substantially improve their trajectory of neurodevelopment. Because children with CP from preterm birth also exhibit a sustained immune system hyper-reactivity, we hypothesized that neuro-immunomodulation with a regimen of repurposed endogenous neurorestorative medications, erythropoietin (EPO) and melatonin (MLT), could improve this trajectory. Thus, we administered EPO + MLT to rats with CP during human infant-toddler equivalency to determine whether we could influence gait patterns in mature animals. After a prenatal injury on embryonic day 18 (E18) that mimics chorioamnionitis at ∼25 weeks human gestation, rat pups were born and raised with their dam. Beginning on postnatal day 15 (P15), equivalent to human infant ∼1 year, rats were randomized to receive either a regimen of EPO + MLT or vehicle (sterile saline) through P20. Gait was assessed in young adult rats at P30 using computerized digital gait analyses including videography on a treadmill. Results indicate that gait metrics of young adult rats treated with an infantile cocktail of EPO + MLT were restored compared to vehicle-treated rats (p < 0.05) and similar to sham controls. These results provide reassuring evidence that pharmacological interventions may be beneficial to infants and toddlers who are diagnosed with CP well after the traditional neonatal window of intervention.

Intraventricular hemorrhage (IVH) occurs in 15-45% of all very low birth weight (VLBW) preterm infants. Despite improvements in the perinatal care, the incidence of IVH remains high. As more preterm infants survive, there will be a larger burden of neurodevelopmental abnormalities borne by former preterm infants.The objective of this study was to develop a predictive clinical model of IVH risk within the first few hours of life in an effort to augment perinatal counseling and guide the timing of future targeted therapies aimed at preventing or slowing the progression of disease.This is a prospective observational cohort study of VLBW infants born in the NICU at John's Hopkins Children's Center from 2011 to 2019. The presence and severity of IVH was defined on standard head ultrasound screening (HUS) using the modified Papile classification. Clinical variables were identified as significant using absolute risk regression from a general linear model. The model predictors included clinically meaningful variables that were not collinear.This study took place at the Johns Hopkins Children's Center Level IV NICU.The study sample included VLBW infants treated in the neonatal intensive care unit (NICU) at John's Hopkins Children's Center from 2011 to 2019. A total of 683 infants included in the study had no or grade I IVH, and 115 infants had grades II through IV IVH. Exclusion criteria included admission to the JHH NICU after 24 h of age, BW gt; 1500 g, and failure to consent.The main outcome of this study was the presence of grades II-IV IVH on standard head ultrasound screening using the modified Papile classification [1].A total of 798 VLBW infants were studied in this cohort and 14.4% had moderate to severe IVH. Fifty four percent of the cohort was black, 33% white, and half of the cohort was male. A higher gestational age, 5-min Apgar score, hematocrit, and platelet count were significantly associated with decreased incidence of IVH in a multi-predictor model (ROC 0.826).In the face of continued lack of treatments for IVH, prevention is still a primary goal to avoid long-term developmental sequela. This model can be used for perinatal counseling and may provide important information during the narrow therapeutic window for targeted prevention therapies.

Neonatal hypoxic ischemic (HI) brain injury causes lifelong neurologic disability. Therapeutic hypothermia (TH) is the only approved therapy that partially mitigates mortality and morbidity. Therapies specifically targeting HI-induced brain cell death are currently lacking. Intracellular calcium dysregulation, oxidative stress, and mitochondrial dysfunction through the formation of the mitochondrial permeability transition pore (mPTP) are drivers of HI cellular injury. GNX-4728, a small molecule direct inhibitor of the mPTP that increases mitochondrial calcium retention capacity, is highly effective in adult neurodegenerative disease models and could have potential as a therapy in neonatal HI. A dose of GNX-4728, equivalent to that used in animal models, 300 mg/kg, IP was highly toxic in p10 mice. We then tested the hypothesis that acute administration of 30 mg/kg, IP of GNX-4728 immediately after HI in a neonatal mouse model would provide neuroprotection. This non-lethal lower dose of GNX-4728 (30 mg/kg, IP) improved the respiratory control ratio of neonatal female HI brain tissue but not in males. Brain injury, assessed histologically with a novel metric approach at 1 and 30 days after HI, was not mitigated by GNX-4728. Our work demonstrates that a small molecule inhibitor of the mPTP has i) an age related toxicity, ii) a sex-related brain mitoprotective profile after HI but iii) this is not sufficient to attenuate forebrain HI neuropathology.

OBJECTIVE: To measure plasma levels of vascular endothelial growth factor (VEGF) and several cytokines (Interleukin [IL]-6 IL-8, IL-10) during the first week of life to examine the relationship between protein expression and likelihood of developing respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). STUDY DESIGN: Levels of IL-6, IL-8, IL-10, and VEGF were measured from plasma obtained from preterm patients during the first week of life. Newborns were recruited from a single center between April 2009 and April 2019. Criteria for the study included being inborn, birth weight of less than 1500 grams, and a gestational age of less than 32 weeks at birth. RESULTS: The development of RDS in preterm newborns was associated with lower levels of VEGF during the first week of life. Higher plasma levels of IL-6 and IL-8 plasma were associated with an increased likelihood and increased severity of BPD at 36 weeks postmenstrual age. In contrast, plasma levels of VEGF, IL-6, IL-8, and IL-10 obtained during the first week of life were not associated with respiratory symptoms and acute care use in young children with BPD in the outpatient setting. CONCLUSIONS: During the first week of life, lower plasma levels of VEGF was associated with the diagnosis of RDS in preterm infants. Preterm infants with higher levels of IL-6 and IL-8 during the first week of life were also more likely to be diagnosed with BPD. These biomarkers may help to predict respiratory morbidities in preterm newborns during their initial hospitalization.

OBJECTIVE To determine if neonatal serum biomarkers representing different pathways of injury differ for cases of HIE of unknown cause to gain insight into timing and mechanism of injury. STUDY DESIGN In this cohort of all neonates with HIE admitted to our NICU, newborns with sentinel events were compared to those without during the 1st 3 days of life. Discard neonatal blood during the 1st 3 days of life was used for analysis. RESULTS Of 277 babies with HIE treated with whole-body hypothermia, 190 (68.6%) had blood available for biomarker analysis. 71 (37.4%) were born within our system, and 119 (62.6%) were transferred in from outside hospitals. Of these babies, 77 (40.5%) had a sentinel event and 113 (59.6%) had no sentinel event. Although the degree of metabolic acidosis was similar, repeated measures analysis showed that during the initial 3 days of life neonates born with HIE in the absence of sentinel events had 41.4% decreased VEGF (p=0.027) and 62.5% increased IL-10 serum concentrations (p=0.005). CONCLUSION These changes indicate that neonatal HIE in the absence of sentinel events is not related to an unrecognized acute intrapartum event and is possibly related to chronic hypoxia of lower severity or recovery from a remote event.

Since 2009, the Neurosciences Intensive Care Nursery at Johns Hopkins Children’s Center has provided a multidisciplinary approach toward the care of newborns with neurological disorders. The program’s cornerstone is an interdisciplinary approach that involves the primary neonatology team plus experts from more than 10 specialties who convene at a weekly team conference at which newborns with neurological problems are discussed in detail. This interdisciplinary approach fosters in-depth discussion of clinical issues to optimize the management of neonates with neurological problems as well as the opportunity to generate research ideas and provide education about neonatal neuroscience at all levels (faculty, nurses, and trainees). The purpose of this article is to provide a 10-year reflection of our Neurosciences Intensive Care Nursery with a view toward expanding efforts in the 3 areas of our mission: clinical care, research, and education. We hope that our experience will enhance the spread of neonatal neuroscience education, care, and research as widely as possible.

BACKGROUND AND PURPOSE Neonatal hypoxic-ischemic injury of the brain and resultant encephalopathy (HIE) leads to major developmental impairments by school age. Conventional/anatomical MRI often fails to detect hippocampal injury in mild cases. We hypothesize that diffusion tensor imaging (DTI) has greater sensitivity for identifying subtle hippocampal injury. METHODS We retrospectively analyzed DTI data collected from a cohort of neonates with HIE and controls. Conventional MRI sequences were classified qualitatively according to severity using a modified Barkovich scale. Using multivariate linear regression, we compared hippocampal DTI scalars of HIE patients and controls. Spearman correlation was used to test the association of DTI scalars in the hippocampal and thalamic regions. A multiple regression analysis tested the association of the DTI scalars with short-term outcomes. RESULTS Fifty-five neonates with HIE (42% males) and 13 controls (54% males) were included. Hippocampal DTI scalars were similar between HIE and control groups, even when restricting the HIE group to those with moderate-to-severe injury (8 subjects). DTI scalars of the thalamus were significantly lower in the moderate-to-severely affected patients compared to controls (right fractional anisotropy [FA] .148 vs. .182, P = .01; left FA .147 vs. .181, P = .03). Hippocampal and thalamic DTI scalars were correlated (P

Therapeutic hypothermia significantly improves survival without disability in near-term and full-term newborns with moderate to severe hypoxic-ischaemic encephalopathy. However, hypothermic neuroprotection is incomplete. The challenge now is to find ways to further improve outcomes. One major limitation to progress is that the specific mechanisms of hypothermia are only partly understood. Evidence supports the concept that therapeutic cooling suppresses multiple extracellular death signals, including intracellular pathways of apoptotic and necrotic cell death and inappropriate microglial activation. Thus, the optimal depth of induced hypothermia is that which effectively suppresses the cell death pathways after hypoxia-ischaemia, but without inhibiting recovery of the cellular environment. Thus mild hypothermia needs to be continued until the cell environment has recovered until it can actively support cell survival. This review highlights that key survival cues likely include the inter-related restoration of neuronal activity and growth factor release. This working model suggests that interventions that target overlapping mechanisms, such as anticonvulsants, are unlikely to materially augment hypothermic neuroprotection. We suggest that further improvements are most likely to be achieved with late interventions that maximise restoration of the normal cell environment after therapeutic hypothermia, such as recombinant human erythropoietin or stem cell therapy.

PURPOSE OF THE REVIEW: The rapid progress in biomarker science is on the threshold of significantly changing clinical care for infants in the Neonatal Intensive Care Unit. Infants with neonatal brain injuries will likely be the first group whose management is dramatically altered with point-of-care, rapidly available brain biomarker analysis. Providing an interim update on progress in this area is the purpose of this review. RECENT FINDINGS: Highlighted findings from the past 18 months of publications on biomarkers in neonatal brain injury include; i) Specific non-brain markers of cardiac health and global asphyxia continue to provide information on brain injury after HIE. ii) Prediction of injury in the piglet HI model is improved with the use of a combination score of plasma metabolites. iii) In a neonatal piglet model of perinatal hypoxia-ischemia, a systemic pro-inflammatory surge of cytokines has been identified after rewarming from therapeutic hypothermia. iv) New biomarkers identified recently include osteopontin, activin A, neutrophil gelatinase-associated lipocalin (NGAL), secretoneurin, Tau, and neurofilament light protein. v) Brain based biomarkers differ in their ability to predict short term in-hospital outcomes and long term neurologic deficits. SUMMARY: Neonatal brain biomarker research is currently in its very early development with major advances still to be made.

The recently developed oscillating-gradient diffusion MRI (OG-dMRI) technique extends our ability to examine brain structures at different spatial scales. In this study, we investigated the sensitivity of OG-dMRI in detecting cellular and subcellular structural changes in a mouse model of neonatal hypoxia ischemia (HI). Neonatal mice received unilateral HI injury or sham injury at postnatal day 10, followed by in vivo T2-weighted and diffusion MRI of the brains at 3–6 h and 24 h after HI. Apparent diffusion coefficient (ADC) maps were acquired using conventional pulsed-gradient dMRI (PG-dMRI) and OG-dMRI with oscillating frequencies from 50 to 200 Hz. Pathology at cellular and subcellular levels was evaluated using neuronal, glial, and mitochondrial markers. We found significantly higher rates of ADC increase with oscillating frequencies (Δ fADC) in the ipsilateral edema region, compared to the contralateral side, starting as early as 3 h after HI. Even in injured regions that showed no apparent change in PG-ADC or pseudo-normalized PG-ADC measurements, Δ fADC remained significantly elevated. Histopathology showed swelling of sub-cellular structures in these regions with no apparent whole-cell level change. These results suggest that OG-dMRI is sensitive to subcellular structural changes in the brain after HI and is less susceptible to pseudo-normalization than PG-dMRI.

Preterm birth is an important cause of perinatal brain injury (PBI). Neurological injury in extremely preterm infants often begins in utero with chorioamnionitis (CHORIO) or inflammation/infection of the placenta and concomitant placental insufficiency. Studies in humans have shown dysregulated inflammatory signaling throughout the placental-fetal brain axis and altered peripheral immune responses in children born preterm with cerebral palsy (CP). We hypothesized that peripheral immune responses would be altered in our well-established rat model of CP. Specifically, we proposed that isolated peripheral blood mononuclear cells (PBMCs) would be hyperresponsive to a second hit of inflammation throughout an extended postnatal time course. Pregnant Sprague-Dawley dams underwent a laparotomy on embryonic day 18 (E18) with occlusion of the uterine arteries (for 60 min) followed by intra-amniotic injection of lipopolysaccharide (LPS, 4 μg/sac) to induce injury in utero. Shams underwent laparotomy only, with equivalent duration of anesthesia. Laparotomies were then closed, and the rat pups were born at E22. PBMCs were isolated from pups on postnatal day 7 (P7) and P21, and subsequently stimulated in vitro with LPS for 3 or 24 h. A secreted inflammatory profile analysis of conditioned media was performed using multiplex electrochemiluminescent immunoassays, and the composition of inflammatory cells was assayed with flow cytometry (FC). Results indicate that CHORIO PBMCs challenged with LPS are hyperreactive and secrete significantly more tumor necrosis factor α (TNFα) and C-X-C chemokine ligand 1 at P7. FC confirmed increased intracellular TNFα in CHORIO pups at P7 following LPS stimulation, in addition to increased numbers of CD11b/c immunopositive myeloid cells. Notably, TNFα secretion was sustained until P21, with increased interleukin 6, concomitant with increased expression of integrin β1, suggesting both sustained peripheral immune hyperreactivity and a heightened activation state. Taken together, these data indicate that in utero injury primes the immune system and augments enhanced inflammatory signaling. The insidious effects of primed peripheral immune cells may compound PBI secondary to CHORIO and/or placental insufficiency, and thereby render the brain susceptible to future chronic neurological disease. Further understanding of inflammatory mechanisms in PBI may yield clinically important biomarkers and facilitate individualized repair strategies and treatments.

Hypoxic-ischemic encephalopathy is a common neonatal brain injury associated with significant morbidity and mortality despite the administration of therapeutic hypothermia (TH). Neonatal seizures and subsequent chronic epilepsy are frequent in this patient population and current treatments are partially effective. We used a neonatal murine hypoxia-ischemia (HI) model to test whether the severity of hippocampal and cortical injury predicts seizure susceptibility 8 days after HI and whether TH mitigates this susceptibility. HI at postnatal day 10 (P10) caused hippocampal injury not mitigated by TH in male or female pups. TH did not confer protection against flurothyl seizure susceptibility at P18 in this model. Hippocampal (R2 = 0.33, p = 0.001) and cortical (R2 = 0.33, p = 0.003) injury directly correlated with seizure susceptibility in male but not female pups. Thus, there are sex-specific consequences of neonatal HI on flurothyl seizure susceptibility in a murine neonatal HI model. Further studies are necessary to elucidate the underlying mechanisms of sex dimorphism in seizure susceptibility after neonatal HI.

Recent technical advances in neurosonography continue broadening the diagnostic utility, sensitivity, and specificity of ultrasound for detecting intracranial abnormalities bed side. The clinical and functional applications of neurosonography have significantly expanded since the 1980s when transcranial Doppler sonography first allowed anatomic and hemodynamic delineation of the intracranial vessels through the thin temporal skull. In the past few years, contrast-enhanced ultrasonography, elastography, 3D/4D reconstruction tools, and high-resolution microvessel imaging techniques have further enhanced the diagnostic significance of neurosonography. Given these advances, a thorough familiarity with these new techniques and devices is crucial for a successful clinical application allowing improved patient care. It is essential that future neurosonography studies compare these advanced techniques against the current "gold standard" computed tomography and magnetic resonance imaging to assure the accuracy of their diagnostic potential. This review will provide a comprehensive update on currently available advanced neurosonography techniques.

Hypoxic-ischemic encephalopathy (HIE) from peripartum complications is a leading cause of neonatal brain injury. Inducing therapeutic hypothermia within 6 hours of birth to 33.0–34.0 ℃ for 72 hours is the standard of care for moderate to severe encephalopathy in many developed countries. The protection afforded by hypothermia is incomplete, however, as nearly half of hypothermia-treated survivors suffer persistent moderate to severe neurologic disabilities (1,2). Therefore, we continue to search for therapeutic strategies that can provide full neuroprotection in all neonates with HIE.

Background Cardiopulmonary injury is common in neonatal encephalopathy, but the link with cerebrovascular dysfunction is unknown. We hypothesized that cerebral autoregulation is associated with cardiopulmonary injury in neonates treated with therapeutic hypothermia (TH) for neonatal encephalopathy. Methods The cerebral hemoglobin volume index (HVx) from near-infrared spectroscopy was used to identify the mean arterial blood pressure (MAP) with optimal autoregulatory vasoreactivity (MAPOPT). We measured associations between MAP relative to MAPOPT and indicators of cardiopulmonary injury (duration of mechanical respiratory support and administration of inhaled nitric oxide (iNO), milrinone, or steroids). Results We identified associations between cerebrovascular autoregulation and cardiopulmonary injury that were often sex-specific. Greater MAP deviation above MAPOPT was associated with shorter duration of intubation in boys but longer ventilatory support in girls. Greater MAP deviation below MAPOPT related to longer intensive care stay in boys. Milrinone was associated with greater MAP deviation below MAPOPT in girls. Conclusion MAP deviation from MAPOPT may relate to cardiopulmonary injury after neonatal encephalopathy, and sex may modulate this relationship. Whereas MAP above MAPOPT may protect the brain and lungs in boys, it may be related to cardiopulmonary injury in girls. Future studies are needed to characterize the role of sex in these associations.

Electroencephalogram (EEG) monitoring techniques for neonatal hypoxia-ischemia (HI) are evolving over time, and the specific type of EEG utilized could influence seizure diagnosis and management. We examined whether the type of EEG performed affected seizure treatment decisions (e.g., the choice and number of antiseizure drugs [ASDs]) in therapeutic hypothermia-treated neonates with HI from 2007 to 2015 in the Johns Hopkins Hospital Neonatal Intensive Care Unit. During this period, 3 different EEG monitoring protocols were utilized: Period 1 (2007-2009), single, brief conventional EEG (1 h duration) at a variable time during therapeutic hypothermia treatment, i.e., ordered when a seizure was suspected; Period 2 (2009-2013), single, brief conventional EEG followed by amplitude-integrated EEG for the duration of therapeutic hypothermia treatment and another brief conventional EEG after rewarming; and Period 3 (2014-2015), continuous video-EEG (cEEG) for the duration of therapeutic hypothermia treatment (72 h) plus for an additional 12 h during and after rewarming. One hundred and sixty-two newborns were included in this retrospective cohort study. As a function of the type and duration of EEG monitoring, we assessed the risk (likelihood) of receiving no ASD, at least 1 ASD, or ≥2 ASDs. We found that the risk of a neonate being prescribed an ASD was 46% less during Period 3 (cEEG) than during Period 1 (brief conventional EEG only) (95% CI 6-69%, p = 0.03). After adjusting for initial EEG and MRI results, compared with Period 1, there was a 38% lower risk of receiving an ASD during Period 2 (95% CI: 9-58%, p = 0.02) and a 67% lower risk during Period 3 (95% CI: 23-86%, p = 0.01). The risk ratio of receiving ≥2 ASDs was not significantly different across the 3 periods. In conclusion, in addition to the higher sensitivity and specificity of continuous video-EEG monitoring, fewer infants are prescribed an ASD when undergoing continuous forms of EEG monitoring (aEEG or cEEG) than those receiving conventional EEG. We recommend that use of continuous video-EEG be considered whenever possible, both to treat seizures more specifically and to avoid overtreatment.

Background: Therapeutic hypothermia provides incomplete neuroprotection for neonatal hypoxic-ischemic encephalopathy (HIE). We examined whether hemodynamic goals that support autoregulation are associated with decreased brain injury and whether these relationships are affected by birth asphyxia or vary by anatomic region. Methods: Neonates cooled for HIE received near-infrared spectroscopy autoregulation monitoring to identify the mean arterial blood pressure with optimized autoregulatory function (MAPOPT). Blood pressure deviation from MAPOPT was correlated with brain injury on MRI after adjusting for the effects of arterial carbon dioxide, vasopressors, seizures, and birth asphyxia severity. Results: Blood pressure deviation from MAPOPT related to neurologic injury in several regions independent of birth asphyxia severity. Greater duration and deviation of blood pressure below MAPOPT were associated with greater injury in the paracentral gyri and white matter. Blood pressure within MAPOPT related to lesser injury in the white matter, putamen and globus pallidus, and brain stem. Finally, blood pressures that exceeded MAPOPT were associated with reduced injury in the paracentral gyri. Conclusions: Blood pressure deviation from optimal autoregulatory vasoreactivity was associated with MRI markers of brain injury that, in many regions, were independent of the initial birth asphyxia. Targeting hemodynamic ranges to optimize autoregulation has potential as an adjunctive therapy to hypothermia for HIE.

We aimed to characterize the parent experience of caring for an infant with neonatal encephalopathy. In this mixed-methods study, we performed semistructured interviews with parents whose infants were enrolled in an existing longitudinal cohort study of therapeutic hypothermia between 2011 and 2014. Thematic saturation was achieved after 20 interviews. Parent experience of caring for a child with neonatal encephalopathy was characterized by 3 principal themes. Theme 1: Many families described cumulative loss and grief throughout the perinatal crisis, critical neonatal course, and subsequent missed developmental milestones. Theme 2: Families experienced entangled infant and broader family interests. Theme 3: Parents evolved into and found meaning in their role as an advocate. These data offer insight into the lived experience of parenting an infant with neonatal encephalopathy. Primary data from parents can serve as a useful framework to guide the development and interpretation of parent-centered outcomes.

The rates of opioid use disorder during pregnancy have more than quadrupled in the last decade, resulting in numerous infants suffering exposure to opioids during the perinatal period, a critical period of central nervous system (CNS) development. Despite increasing use, the characterization and definition of the molecular and cellular mechanisms of the long-term neurodevelopmental impacts of opioid exposure commencing in utero remains incomplete. Thus, in consideration of the looming public health crisis stemming from the multitude of infants with prenatal opioid exposure entering school age, we undertook an investigation of the effects of perinatal methadone exposure in a novel preclinical model. Specifically, we examined the effects of opioids on the developing brain to elucidate mechanisms of putative neural cell injury, to identify diagnostic biomarkers and to guide clinical studies of outcome and follow-up. We hypothesized that methadone would induce a pronounced inflammatory profile in both dams and their pups, and be associated with immune system dysfunction, sustained CNS injury, and altered cognition and executive function into adulthood. This investigation was conducted using a combination of cellular, molecular, biochemical, and clinically translatable biomarker, imaging and cognitive assessment platforms. Data reveal that perinatal methadone exposure increases inflammatory cytokines in the neonatal peripheral circulation, and reprograms and primes the immune system through sustained peripheral immune hyperreactivity. In the brain, perinatal methadone exposure not only increases chemokines and cytokines throughout a crucial developmental period, but also alters microglia morphology consistent with activation, and upregulates TLR4 and MyD88 mRNA. This increase in neuroinflammation coincides with reduced myelin basic protein and altered neurofilament expression, as well as reduced structural coherence and significantly decreased fractional anisotropy on diffusion tensor imaging. In addition to this microstructural brain injury, adult rats exposed to methadone in the perinatal period have significant impairment in associative learning and executive control as assessed using touchscreen technology. Collectively, these data reveal a distinct systemic and neuroinflammatory signature associated with prenatal methadone exposure, suggestive of an altered CNS microenvironment, dysregulated developmental homeostasis, complex concurrent neural injury, and imaging and cognitive findings consistent with clinical literature. Further investigation is required to define appropriate therapies targeted at the neural injury and improve the long-term outcomes for this exceedingly vulnerable patient population.

Early studies following perinatal hypoxic-ischemic encephalopathy (HIE) suggested expressive language deficits and academic difficulties, but there is only limited detailed study of language development in this population since the widespread adoption of therapeutic hypothermia (TH). Expressive and receptive language testing was performed as part of a larger battery with 45 children with a mean age of 26 months following perinatal HIE treated with TH. Overall cohort outcomes as well as the effects of gender, estimated household income, initial pH and base excess, and pattern of injury on neonatal brain MRI were assessed. The cohort overall demonstrated expressive language subscore, visual-reception subscore, and early learning composite scores significantly below test norms, with relative sparing of receptive language subscores. Poorer expressive language manifested as decreased vocabulary size and shorter utterances. Expressive language subscores showed a significant gender effect, and estimated socioeconomic status showed a significant effect on both receptive and expressive language subscores. Initial blood gas markers and modified Sarnat scoring did not show a significant effect on language subscores. Binarized MRI abnormality predicted a significant effect on both receptive and expressive language subscores; the presence of specific cortical/subcortical abnormalities predicted receptive language deficits. Overall, the language development profile of children following HIE in the era of hypothermia shows a relative strength in receptive language. Gender and socioeconomic status predominantly predict expressive language deficits; abnormalities detectable on MRI predominantly predict receptive language deficits.

Aim: Hypoxic-ischemic encephalopathy is associated with damage to deep gray matter; however, white matter involvement has become recognized. This study explored differences between patients and clinical controls on diffusion tensor imaging, and relationships between diffusion tensor imaging and neurodevelopmental outcomes. Method: Diffusion tensor imaging was obtained for 31 neonates after hypoxic-ischemic encephalopathy treated with therapeutic hypothermia and 10 clinical controls. A subgroup of patients with hypoxic-ischemic encephalopathy (n = 14) had neurodevelopmental outcomes correlated with diffusion tensor imaging scalars. Results: Group differences in diffusion tensor imaging scalars were observed in the putamen, anterior and posterior centrum semiovale, and the splenium of the corpus callosum. Differences in these regions of interest were correlated with neurodevelopmental outcomes between ages 20 and 32 months. Conclusion: Therapeutic hypothermia may not be a complete intervention for hypoxic-ischemic encephalopathy, as neonatal white matter changes may continue to be evident, but further research is warranted. Patterns of white matter change on neonatal diffusion tensor imaging correlated with neurodevelopmental outcomes in this exploratory pilot study.

Background Hypoxic-ischemic injury (HII) is a major cause of neonatal death and neurodevelopmental disability. Head ultrasounds (HUS) in neonates with HII often show enhanced gray/white matter differentiation. We assessed the significance of this finding in predicting white matter structural integrity measured by diffusion tensor imaging (DTI) in neonates with HII. Methods We performed a quantitative region of interest-based analysis of white and gray matter echogenicity within the cingulate gyrus on pre- and posthypothermia HUS. We also completed a quantitative analysis of fractional anisotropy (FA) and mean (MD), axial (AD), and radial (RD) diffusivity within the bilateral anterior and posterior centrum semiovale (CSO) on posthypothermia brain magnetic resonance imaging. For HUS studies, we calculated a white-to-gray matter echogenicity ratio (WGR) and subsequently correlated it to DTI measurements. Results Forty-two term neonates with HII who underwent hypothermia therapy were included. Significant correlation was found between prehypothermia WGR and MD, AD, and RD values in the left anterior CSO (r = .38-.40, P = .02). Prehypothermia WGR also correlated with the following: MD and RD in the right anterior CSO (r = .35-.36, P = .04), MD and AD in the right posterior CSO (r = .32-.45, P = .008-.03), and AD in the left posterior CSO (r = .47, P = .005). No significant correlation was found either between prehypothermia WGR and FA values in the bilateral anterior and posterior CSO or between posthypothermia WGR and all DTI scalars in the bilateral anterior and posterior CSO. Conclusions Prehypothermia HUS WGR may predict posthypothermia white matter structural integrity and is potentially an early and easily obtainable biomarker of severity in neonatal HII.

Since neonatal hypoxia-ischemia (HI) disrupts the hippocampal (Hp) GABAergic network in the mouse and Hp injury in this model correlates with flurothyl seizure susceptibility only in male mice, we hypothesized that GABAergic disruption correlates with flurothyl seizure susceptibility in a sex-specific manner. C57BL6 mice were exposed to HI (Vannucci model) versus sham procedures at P10, randomized to normothermia (NT) or therapeutic hypothermia (TH), and subsequently underwent flurothyl seizure testing at P18. Only in male mice, Hp atrophy correlated with seizure susceptibility. The number of Hp parvalbumin positive interneurons (PV+INs) decreased after HI in both sexes, but TH attenuated this deficit only in females. In males only, seizure susceptibility directly correlated with the number of PV+INs, but not somatostatin or calretinin expressing INs. Hp GABAB receptor subunit levels were decreased after HI, but unrelated to later seizure susceptibility. In contrast, Hp GABAA receptor α1 subunit (GABAARα1) levels were increased after HI. Adjusting the number of PV+ INs for their GABAARα1 expression strengthened the correlation with seizure susceptibility in male mice. Thus, we identified a novel Hp sex-specific GABA-mediated mechanism of compensation after HI that correlates with flurothyl seizure susceptibility warranting further study to better understand potential clinical translation.

Objectives To identify candidate biomarkers in both plasma and cerebrospinal fluid (CSF) that are associated with neonatal encephalopathy severity measured by encephalopathy grade, seizures, brain injury by magnetic resonance imaging (MRI), and neurodevelopmental outcomes at 15-30 months. Study design A retrospective cohort study of plasma (N = 155, day of life 0-1) and CSF (n = 30, day of life 0-7) from neonates with neonatal encephalopathy and healthy neonates born at term (N = 30, ≥36 weeks of gestation) was conducted. We measured central nervous system necrosis (glial fibrillary acidic protein [GFAP], neurogranin [NRGN], tau), inflammatory (interleukin [IL]-6, IL-8, IL-10), and trophic (brain-derived neurotrophic factor [BDNF], vascular endothelial growth factor) proteins. Clinical outcomes were Sarnat scores of encephalopathy, seizures, MRI scores, and Bayley Scales of Infant and Toddler Development III at 15-30 months. Results Plasma NRGN, tau, IL-6, IL-8, and IL-10 were greater, whereas BDNF and vascular endothelial growth factor were lower in patients with neonatal encephalopathy vs controls. In plasma, tau, GFAP, and NRGN were directly and BDNF inversely associated with encephalopathy grade. IL-6 was inversely related to seizures. Tau was directly related to MRI abnormalities. Tau was inversely associated with Bayley Scales of Infant and Toddler Development III cognitive and motor outcomes. In CSF, NRGN was inversely associated with cognitive, motor, and language measures. GFAP, IL-6, and IL-10 were inversely related to cognitive and motor outcomes. IL-8 was inversely related to motor outcomes. CSF candidate biomarkers showed no significant relationships with encephalopathy grade, seizures, or MRI abnormalities. Conclusions Plasma candidate biomarkers predicted encephalopathy severity, seizures, MRI abnormalities, and neurodevelopmental outcomes at 15-30 months.

Toll-like receptor (TLR)3 activation during the neonatal period produces responses linked to the origins of neuropsychiatric disorders. Although there is sexual dimorphism in neuropsychiatric disorders, it is unknown if brain responses to TLR3 activation are sex-specific. We hypothesized that poly I:C in a post-natal day (P)8 model induces a sexually dimorphic inflammatory responses. C57BL6 mice received intraperitoneal injection of poly I:C (10 mg/kg) or vehicle [normal saline (NS)] at P8. Pups were killed at 6 or 14 h for caspase 3 and 8 activity assays, NFkB ELISA, IRF3, AP1, and GFAP western blotting and cytokines/chemokines gene expression real time qRT-PCR (4–6/group). A second group of pups were killed at 24 h (P9) or 7 days (P15) after poly I:C to assess astrocytic (GFAP) and microglia (Iba1) activation in the hippocampus, thalamus and cortex using immunohistochemistry, and gene and protein expression of cytokines/chemokines using real time RT-PCR and MSD, respectively (4–6/group). Non-parametric analysis was applied. Six hours after poly I:C, caspase-3 and -8 activities in cytosolic fractions were 1.6 and 2.8-fold higher in poly I:C-treated than in NS-treated female mice, respectively, while gene expressions of pro-inflammatory cytokines were upregulated in both sexes. After poly I:C, IRF3 nuclear translocation occurred earlier (6 h) in female mice and later (14 h) in male mice. At 14 h after poly I:C, only male mice also had increased nuclear NFκB levels (88%, p < 0.001) and GFAP expression coinciding with persistent IL-6 and FAS gene upregulation (110 and 77%, respectively; p < 0.001) and IL-10 gene downregulation (-42%, p < 0.05). At 24 h after poly I:C, IL-1β, CXCL-10, TNF-α, and MCP-1 were similarly increased in both sexes but at 7 days after exposure, CXCL-10 and INFγ were increased and IL-10 was decreased only in female mice. Accordingly, microglial activation persisted at 7 days after poly I:C in the hippocampus, thalamus and cortex of female mice. This preliminary study suggests that TLR3 activation may produce in the developing neonatal mouse brain a sexually dimorphic response with early activation of caspase-dependent pathways in female mice, activation of inflammatory cascades in both sexes, which then persists in female mice. Further well-powered studies are essential to confirm these sex-specific findings.

BACKGROUND AND PURPOSE: Neonatal hypoxic-ischemic injury of the brain and resultant encephalopathy (HIE) leads to major developmental impairments by school age. Conventional/anatomical MRI often fails to detect hippocampal injury in mild cases. We hypothesize that diffusion tensor imaging (DTI) has greater sensitivity for identifying subtle hippocampal injury. METHODS: We retrospectively analyzed DTI data collected from a cohort of neonates with HIE and controls. Conventional MRI sequences were classified qualitatively according to severity using a modified Barkovich scale. Using multivariate linear regression, we compared hippocampal DTI scalars of HIE patients and controls. Spearman correlation was used to test the association of DTI scalars in the hippocampal and thalamic regions. A multiple regression analysis tested the association of the DTI scalars with short-term outcomes. RESULTS: Fifty-five neonates with HIE (42% males) and thirteen controls (54% males) were included. Hippocampal DTI scalars were similar between HIE and control groups, even when restricting the HIE group to those with moderate-to-severe injury (8 subjects). DTI scalars of the thalamus were significantly lower in the moderate-to-severely affected patients compared to controls (right FA 0.148 vs. 0.182, p=0.01; left FA 0.147 vs. 0.181, p=0.03). Hippocampal and thalamic DTI scalars were correlated (p

Background Deviation of mean arterial blood pressure (MAP) from the range that optimizes cerebral autoregulatory vasoreactivity (optimal MAP) could increase neurological injury from hypoxic-ischemic encephalopathy (HIE). We tested whether a global magnetic resonance imaging (MRI) brain injury score and regional diffusion tensor imaging (DTI) are associated with optimal MAP in neonates with HIE. Methods Twenty-five neonates cooled for HIE were monitored with the hemoglobin volume index. In this observational study, we identified optimal MAP and measured brain injury by qualitative and quantitative MRIs with the Neonatal Research Network (NRN) score and DTI mean diffusivity scalars. Optimal MAP and blood pressure were compared with brain injury. Results Neonates with blood pressure measurements within optimal MAP during rewarming had less brain injury by NRN score ( P = 0.040). Longer duration of MAP within optimal MAP during hypothermia correlated with higher mean diffusivity in the anterior centrum semiovale ( P = 0.008) and pons ( P = 0.002). Blood pressure deviation below optimal MAP was associated with lower mean diffusivity in cerebellar white matter ( P = 0.033). Higher optimal MAP values related to lower mean diffusivity in the basal ganglia ( P = 0.021), the thalamus ( P = 0.006), the posterior limb of the internal capsule ( P = 0.018), the posterior centrum semiovale ( P = 0.035), and the cerebellar white matter ( P = 0.008). Optimal MAP values were not associated with the NRN score. Conclusions The NRN score and the regional mean diffusivity scalars detected injury with mean arterial blood pressure deviations from the optimal MAP. Higher optimal MAP and lower mean diffusivity may be related because of cytotoxic edema and limited vasodilatory reserve at low MAP in injured brain. DTI detected injury with elevated optimal MAP better than the NRN score.