Search

Background: Intestinal fatty acid binding protein (I-FABP) is an intestinal epithelial protein detectable in infants with necrotizing enterocolitis (NEC). The longitudinal behavior of I-FABP following NEC or its association with gastrointestinal or neurodevelopmental outcomes is unknown., Methods: In this secondary analysis of the Preterm Erythropoietin Neuroprotection Trial, we compared infants with and without NEC. Urine I-FABP concentrations in matched infants (n = 70) were measured serially using ELISA and compared using paired analysis. In infants with NEC, the associations of I-FABP levels with short-term outcomes and neurodevelopmental outcomes at 22-26 months corrected age were determined using non-parametric analysis., Results: Infants with NEC were more likely to have cholestasis, death or severe neurodevelopmental impairment, cerebral palsy, and lower Bayley-III motor scores. Baseline urinary I-FABP levels were similar between groups. When compared to controls, infants with NEC had urinary I-FABP concentrations that were higher at diagnosis (median 11 vs 2.6 ng/ml, p = 0.006) and lower post-NEC (median 1 vs 5 ng/ml, p = 0.002). Diagnosis I-FABP levels were not associated with gastrointestinal or neurodevelopmental outcomes at 22-26 months corrected age., Conclusions: In extremely preterm infants, urinary I-FABP was elevated at NEC diagnosis and lower post-NEC compared to matched controls. I-FABP levels were not associated with adverse gastrointestinal or neurodevelopmental outcomes., Impact: Urinary intestinal fatty acid binding protein (I-FABP) levels are increased at diagnosis of NEC and fall to below baseline after NEC in extremely preterm infants. Urine I-FABP levels at NEC diagnosis are not associated with cholestasis, intestinal stricture or obstruction, need for additional intestinal surgery after NEC, or neurodevelopmental outcomes at 22-26 months corrected age. Urine I-FABP levels may be useful in the diagnosis of NEC. Diagnostic I-FABP levels do not predict short-term gastrointestinal or neurodevelopmental outcomes after NEC., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Objective: To determine whether an enteral, clonidine-based sedation strategy (CLON) during therapeutic hypothermia (TH) for hypoxic-ischemic encephalopathy would decrease opiate use while maintaining similar short-term safety and efficacy profiles to a morphine-based strategy (MOR)., Study Design: This was a single-center, observational study conducted at a level IV neonatal intensive care unit from January 1, 2017, to October 1, 2021. From April 13, 2020, to August 13, 2020, we transitioned from MOR to CLON. Thus, patients receiving TH for hypoxic-ischemic encephalopathy were grouped to MOR (before April 13, 2020) and CLON (after August 13, 2020). We calculated the total and rescue morphine milligram equivalent/kg (primary outcome) and frequency of hemodynamic changes (secondary outcome) for both groups., Results: The MOR and CLON groups (74 and 25 neonates, respectively) had similar baseline characteristics and need for rescue sedative intravenous infusion (21.6% MOR and 20% CLON). Both morphine milligram equivalent/kg and need for rescue opiates (combined bolus and infusions) were greater in MOR than CLON (P < .001). As days in TH advanced, a lower percentage of patients receiving CLON needed rescue opiates (92% on day 1 to 68% on day 3). Patients receiving MOR received a greater cumulative dose of dopamine and more frequently required a second inotrope and hydrocortisone for hypotension. MOR had a lower respiratory rate during TH (P = .01 vs CLON)., Conclusions: Our CLON protocol is noninferior to MOR, maintaining perceived effectiveness and hemodynamic safety, with an apparently reduced need for opiates and inotropes., Competing Interests: Declaration of Competing Interest Supported by National Institutes of Health (KO8NS096115 [R.C.-V.], R01NS126549, R01 HD110091, R21NS123814 [R.C.-V. and F.N.]), the Thomas Wilson Foundation [R.C.-V.], and JHU Department of Pediatrics Innovation Grant [F.N. and R.C.-V.]. The authors have no conflicts of interest relevant to this article to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hypoxic-ischemic encephalopathy (HIE) in neonates causes mortality and neurologic morbidity, including poor cognition with a complex neuropathology. Injury to the cholinergic basal forebrain and its rich innervation of cerebral cortex may also drive cognitive pathology. It is uncertain whether genes associated with adult cognition-related neurodegeneration worsen outcomes after neonatal HIE. We hypothesized that neocortical damage caused by neonatal HI in mice is ushered by persistent cholinergic innervation and interneuron (IN) pathology that correlates with cognitive outcome and is exacerbated by genes linked to Alzheimer's disease. We subjected non-transgenic (nTg) C57Bl6 mice and mice transgenically (Tg) expressing human mutant amyloid precursor protein (APP-Swedish variant) and mutant presenilin (PS1-ΔE9) to the Rice-Vannucci HI model on postnatal day 10 (P10). nTg and Tg mice with sham procedure were controls. Visual discrimination (VD) was tested for cognition. Cortical and hippocampal cholinergic axonal and IN pathology and Aβ plaques, identified by immunohistochemistry for choline acetyltransferase (ChAT) and 6E10 antibody respectively, were counted at P210. Simple ChAT+ axonal swellings were present in all sham and HI groups; Tg mice had more than their nTg counterparts, but HI did not affect the number of axonal swellings in APP/PS1 Tg mice. In contrast, complex ChAT+ neuritic clusters (NC) occurred only in Tg mice; HI increased that burden. The abundance of ChAT+ clusters in specific regions correlated with decreased VD. The frequency of attritional ChAT+ INs in the entorhinal cortex (EC) was increased in Tg shams relative to their nTg counterparts, but HI obviated this difference. Cholinergic IN pathology in EC correlated with NC number. The Aβ deposition in APP/PS1 Tg mice was not exacerbated by HI, nor did it correlate with other metrics. Adult APP/PS1 Tg mice have significant cortical cholinergic axon and EC ChAT+ IN pathologies; some pathology was exacerbated by neonatal HI and correlated with VD. Mechanisms of neonatal HI induced cognitive deficits and cortical neuropathology may be modulated by genetic risk, perhaps accounting for some of the variability in outcomes., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Neonatal hypoxic-ischemic encephalopathy (HIE) is the leading cause of neurodevelopmental morbidity in term infants worldwide. Incidence of HIE is highest in low and middle-income communities with minimal access to neonatal intensive care and an underdeveloped infrastructure for advanced neurologic interventions. Moreover, therapeutic hypothermia, standard of care for HIE in high resourced settings, is shown to be ineffective in low and middle-income communities. With their low cost, ease of administration, and capacity to potently target the central nervous system, intranasal therapies pose a unique opportunity to be a more globally accessible treatment for neonatal HIE. Intranasal experimental therapeutics have been studied in both rodent and piglet models, but no intranasal therapeutics for neonatal HIE have undergone human clinical trials. Additional research must be done to expand the array of treatments available for use as intranasal therapies for neonatal HIE thus improving the neurologic outcomes of infants worldwide.

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of histone H3 lysine 26 trimethylation (H3K36me3) at the SP1 locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5-regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events., Competing Interests: Declaration of interests H.T.B. is a consultant for Mahzi Therapeutics and founder of Kaldur Therapeutics. S.R. and H.T.B. have a European patent application (23167505.9) on a therapeutic strategy to activate the MHR., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Noninvasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent advances in diffusion magnetic resonance imaging enabled in vivo examination of tissue microstructures well beyond the imaging resolution. Here, we proposed to use diffusion time-dependent diffusion kurtosis imaging ( t DKI) to simultaneously assess cellular morphology and transmembrane permeability in hypoxic-ischemic (HI) brain injury. Through numerical simulations and organoid imaging, we demonstrated the feasibility of capturing effective size and permeability changes using t DKI. In vivo MRI of HI-injured mouse brains detected a shift of the t DKI peak to longer diffusion times, suggesting swelling of the cellular processes. Furthermore, we observed a faster decrease of the t DKI tail, reflecting increased transmembrane permeability associated with up-regulated water exchange or necrosis. Such information, unavailable from a single diffusion time, can predict salvageable tissues. Preliminary applications of t DKI in patients with ischemic stroke suggested increased transmembrane permeability in stroke regions, illustrating t DKI's potential for detecting pathological changes in the clinics.

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of H3K36me3 at the SP1 -locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5 regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events., Competing Interests: Competing interests: HTB is a consultant for Mahzi Therapeutics and founder of Kaldur Therapeutics. SR and HTB have a European patent application (#23167505.9) on a therapeutic strategy to activate the MHR. Other authors declare that they have no competing interests.

Quantitative analysis of electroencephalography (qEEG) is a potential source of biomarkers for neonatal encephalopathy (NE). However, prior studies using qEEG in NE were limited in their generalizability due to individualized techniques for calculating qEEG features or labor-intensive pre-selection of EEG data. We piloted a fully automated method using commercially available software to calculate the suppression ratio (SR), absolute delta power, and relative delta, theta, alpha, and beta power from EEG of neonates undergoing 72 h of therapeutic hypothermia (TH) for NE between April 20, 2018, and November 4, 2019. We investigated the association of qEEG with degree of encephalopathy (modified Sarnat score), severity of neuroimaging abnormalities following TH (National Institutes of Child Health and Development Neonatal Research Network [NICHD-NRN] score), and presence of seizures. Thirty out of 38 patients met inclusion criteria. A more severe modified Sarnat score was associated with higher SR during all phases of TH, lower absolute delta power during all phases except rewarming, and lower relative delta power during the last 24 h of TH. In 21 patients with neuroimaging data, a worse NICHD-NRN score was associated with higher SR, lower absolute delta power, and higher relative beta power during all phases. QEEG features were not significantly associated with the presence of seizures after correction for multiple comparisons. Our results are consistent with those of prior studies using qEEG in NE and support automated qEEG analysis as an accessible, generalizable method for generating biomarkers of NE and response to TH. Additionally, we found evidence of an immature relative frequency composition in neonates with more severe brain injury, suggesting that automated qEEG analysis may have a use in the assessment of brain maturity., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Neonatal hypoxic-ischemic encephalopathy (HIE) is the leading cause of acquired neonatal brain injury with the risk of developing serious neurological sequelae and death. An accurate and robust prediction of short- and long-term outcomes may provide clinicians and families with fundamental evidence for their decision-making, the design of treatment strategies, and the discussion of developmental intervention plans after discharge. Diffusion tensor imaging (DTI) is one of the most powerful neuroimaging tools with which to predict the prognosis of neonatal HIE by providing microscopic features that cannot be assessed by conventional magnetic resonance imaging (MRI). DTI provides various scalar measures that represent the properties of the tissue, such as fractional anisotropy (FA) and mean diffusivity (MD). Since the characteristics of the diffusion of water molecules represented by these measures are affected by the microscopic cellular and extracellular environment, such as the orientation of structural components and cell density, they are often used to study the normal developmental trajectory of the brain and as indicators of various tissue damage, including HIE-related pathologies, such as cytotoxic edema, vascular edema, inflammation, cell death, and Wallerian degeneration. Previous studies have demonstrated widespread alteration in DTI measurements in severe cases of HIE and more localized changes in neonates with mild-to-moderate HIE. In an attempt to establish cutoff values to predict the occurrence of neurological sequelae, MD and FA measurements in the corpus callosum, thalamus, basal ganglia, corticospinal tract, and frontal white matter have proven to have an excellent ability to predict severe neurological outcomes. In addition, a recent study has suggested that a data-driven, unbiased approach using machine learning techniques on features obtained from whole-brain image quantification may accurately predict the prognosis of HIE, including for mild-to-moderate cases. Further efforts are needed to overcome current challenges, such as MRI infrastructure, diffusion modeling methods, and data harmonization for clinical application. In addition, external validation of predictive models is essential for clinical application of DTI to prognostication., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Background: Extremely low birth weight (ELBW) infants comprise a fragile population at risk for neurodevelopmental disabilities (NDD). Systemic steroids were previously associated with NDD, but more recent studies suggest hydrocortisone (HCT) may improve survival without increasing NDD. However, the effects of HCT on head growth adjusted for illness severity during NICU hospitalization are unknown. Thus, we hypothesize that HCT will protect head growth, accounting for illness severity using a modified neonatal Sequential Organ Failure Assessment (M-nSOFA) score., Methods: We conducted a retrospective study that included infants born at 23-29 weeks gestational age (GA) and < 1000 g. Our study included 73 infants, 41% of whom received HCT., Results: We found negative correlations between growth parameters and age, similar between HCT and control patients. HCT-exposed infants had lower GA but similar normalized birth weights; HCT-exposed infants also had higher illness severity and longer lengths of hospital stay. We found an interaction between HCT exposure and illness severity on head growth, such that infants exposed to HCT had better head growth compared to those not exposed to HCT when adjusted for illness severity., Conclusion: These findings emphasize the importance of considering patient illness severity and suggest that HCT use may offer additional benefits not previously considered., Impact: This is the first study to assess the relationship between head growth and illness severity in extremely preterm infants with extremely low birth weights during their initial NICU hospitalization. Infants exposed to hydrocortisone (HCT) were overall more ill than those not exposed, yet HCT exposed infants had better preserved head growth relative to illness severity. Better understanding of the effects of HCT exposure on this vulnerable population will help guide more informed decisions on the relative risks and benefits for HCT use., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Non-invasive mapping of cellular pathology can provide critical diagnostic and prognostic information. Recent developments in diffusion MRI have produced new tools for examining tissue microstructure at a level well below the imaging resolution. Here, we report the use of diffusion time ( t )-dependent diffusion kurtosis imaging ( t DKI) to simultaneously assess the morphology and transmembrane permeability of cells and their processes in the context of pathological changes in hypoxic-ischemic brain (HI) injury. Through Monte Carlo simulations and cell culture organoid imaging, we demonstrate feasibility in measuring effective size and permeability changes based on the peak and tail of t DKI curves. In a mouse model of HI, in vivo imaging at 11.7T detects a marked shift of the t DKI peak to longer t in brain edema, suggesting swelling and beading associated with the astrocytic processes and neuronal neurites. Furthermore, we observed a faster decrease of the t DKI tail in injured brain regions, reflecting increased membrane permeability that was associated with upregulated water exchange upon astrocyte activation at acute stage as well as necrosis with disrupted membrane integrity at subacute stage. Such information, unavailable with conventional diffusion MRI at a single t, can predict salvageable tissues. For a proof-of-concept, t DKI at 3T on an ischemic stroke patient suggested increased membrane permeability in the stroke region. This work therefore demonstrates the potential of t DKI for in vivo detection of the pathological changes in microstructural morphology and transmembrane permeability after ischemic injury using a clinically translatable protocol.

Importance: 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., Objective: 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., Design: 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., Setting: This study took place at the Johns Hopkins Children's Center Level IV NICU., Participants: 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., Main Outcome: 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]., Results: 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)., Conclusion and Relevance: 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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hypoxic-ischemic encephalopathy (HIE) is the most common cause of neonatal acquired brain injury. Although conventional MRI may predict neurodevelopmental outcomes, accurate prognostication remains difficult. As diffusion tensor imaging (DTI) may provide an additional diagnostic and prognostic value over conventional MRI, we aimed to develop a composite DTI (cDTI) score to relate to short-term neurological function. Sixty prospective neonates treated with therapeutic hypothermia (TH) for HIE were evaluated with DTI, with a voxel size of 1 × 1 × 2 mm. Fractional anisotropy (FA) and mean diffusivity (MD) from 100 neuroanatomical regions (FA/MD * 100 = 200 DTI parameters in total) were quantified using an atlas-based image parcellation technique. A least absolute shrinkage and selection operator (LASSO) regression was applied to the DTI parameters to generate the cDTI score. Time to full oral nutrition [short-term oral feeding (STO) score] was used as a measure of short-term neurological function and was correlated with extracted DTI features. Seventeen DTI parameters were selected with LASSO and built into the final unbiased regression model. The selected factors included FA or MD values of the limbic structures, the corticospinal tract, and the frontotemporal cortices. While the cDTI score strongly correlated with the STO score (rho = 0.83, p = 2.8 × 10 -16 ), it only weakly correlated with the Sarnat score (rho = 0.27, p = 0.035) and moderately with the NICHD-NRN neuroimaging score (rho = 0.43, p = 6.6 × 10 -04 ). In contrast to the cDTI score, the NICHD-NRN score only moderately correlated with the STO score (rho = 0.37, p = 0.0037). Using a mixed-model analysis, interleukin-10 at admission to the NICU (p = 1.5 × 10 -13 ) and tau protein at the end of TH/rewarming ( p = 0.036) and after rewarming ( p = 0.0015) were significantly associated with higher cDTI scores, suggesting that high cDTI scores were related to the intensity of the early inflammatory response and the severity of neuronal impairment after TH. In conclusion, a data-driven unbiased approach was applied to identify anatomical structures associated with some aspects of neurological function of HIE neonates after cooling and to build a cDTI score, which was correlated with the severity of short-term neurological functions., Competing Interests: KOi is a consultant for “AnatomyWorks” and “Corporate-M.” This arrangement is being managed by the Johns Hopkins University in accordance with its conflict-of-interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Onda, Catenaccio, Chotiyanonta, Chavez-Valdez, Meoded, Soares, Tekes, Spahic, Miller, Parker, Parkinson, Vaidya, Graham, Stafstrom, Everett, Northington and Oishi.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2021 Wiley Periodicals LLC.)

Objective: To determine a safe dose of clonidine (CLON) to be used in infants with hypoxic ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH)., Study Design: 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)., Results: The CLON group received less "as needed" MOR than the MOR-only group for agitation/shivering (p < 0.001), and the CLON vs. MOR-only group spent 92% vs. 79% of cooling time at the target core body temperature (CBT; p = 0.03, CLON vs. MOR)., Conclusions: Intravenous CLON (1 mcg/kg Q8h) is well tolerated in infants treated with TH for HIE. CLON stabilizes CBT in the ideal range during cooling, which may be optimal for neuroprotection., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (© 2022 S. Karger AG, Basel.)

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., (© 2022 S. Karger AG, Basel.)

Objective: To determine the changes due to therapeutic hypothermia (TH) exposure in the strength of association between traditional clinical and biochemical indicators of severity of neonatal hypoxic-ischemic encephalopathy (HIE) and serum biomarkers. We hypothesized that culmination of TH changes the strength of the relationships between traditional indicators of severity of HIE and serum biomarkers. Methods: This was a single-center observational cohort study of 178 neonates with HIE treated with TH and followed with serum biomarkers: (i) brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) (neurotrophins); (ii) tau and glial fibrillary acidic protein (GFAP) (neural cell injury); and (iii) interleukin 6 (IL-6), IL-8, and IL-10 (cytokines), during their first week of life. Adjusted mixed-effect models tested associations with HIE indicators in relation to TH exposure. Results: At admission, lower Apgar scores and base excess (BE) and higher lactate and nucleated red blood cell (NRBC) count correlated with higher Sarnat scores. These indicators of worse HIE severity, including higher Sarnat score, correlated with lower VEGF and higher tau, GFAP, and IL-10 levels at different time points. Within the first 24 h of life, patients with a Sarnat score >2 had lower VEGF levels, whereas only those with score of 3 also had higher GFAP and IL-10 levels. Tau levels increased during TH in patients with Sarnat score of 3, whereas tau and GFAP increased after TH in those with scores of 2. After adjustments, lower VEGF levels during TH and higher tau, GFAP, and IL-10 levels during and after TH were associated with worse Sarnat scores. Tau and GFAP relationship with Sarnat score became stronger after TH. Conclusion: Therapeutic hypothermia exerts an independent modulatory effect in the relationships between traditional indicators of severity of HIE and serum biomarkers after adjustments. Thus, the timing of biomarker testing in relation to TH exposure must be carefully considered if biomarkers are proposed for patient stratification in novel clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chavez-Valdez, Miller, Spahic, Vaidya, Parkinson, Dietrick, Brooks, Gerner, Tekes, Graham, Northington and Everett.)

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., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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. In total, 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.

Neonatal hypoxia-ischemia (nHI) disrupts hippocampal GABAergic development leading to memory deficits in mice. Polysialic-acid neural-cell adhesion molecule (PSA-NCAM) developmentally declines to trigger GABAergic maturation. We hypothesized that nHI changes PSA-NCAM abundance and cellular distribution, impairing GABAergic development, and marking nascent neurodegeneration. Cell degeneration, atrophy, and PSA-NCAM immunoreactivity (IR) were measured in CA1 of nHI-injured C57BL6 mice related to: (i) cellular subtype markers; (ii) GAD65/67 and synatophysin (SYP), pre-synaptic markers; (iii) phospho-Ser 396 Tau, cytoskeletal marker; and (iv) GAP43, axonalregeneration marker. PSA-NCAM IR was minimal in CA1 of shams at P11. After nHI, PSA-NCAM IR was increased in injured pyramidal cells (PCs), minimal in parvalbumin (PV) + INs, and absent in glia. PSA-NCAM IR correlated with injury severity and became prominent in perikaryal cytoplasm at P18. GAD65/67 and SYP IRs only weakly related to PSA-NCAM after nHI. Injured phospho-Ser 396 Tau + PCs and PV + INs variably co-expressed PSA-NCAM at P40. While PCs with cytoplasmic marginalized PSA-NCAM had increased perisomatic GAP43, those with perikaryal cytoplasmic PSA-NCAM had minimal GAP43. PSA-NCAM increased in serum of nHI-injured mice. Increased PSA-NCAM is likely a generic acute response to nHI brain injury. PSA-NCAM aberrant cellular localization may aggravate neuronal degeneration. The significance of PSA-NCAM as a biomarker of recovery from nHI and nascent neurodegeneration needs further study.

Dysfunctional cerebrovascular autoregulation may contribute to neurologic injury in neonatal hypoxic-ischemic encephalopathy (HIE). Identifying the optimal mean arterial blood pressure (MAPopt) that best supports autoregulation could help identify hemodynamic goals that support neurologic recovery. In neonates who received therapeutic hypothermia for HIE, we hypothesized that the wavelet hemoglobin volume index (wHVx) would identify MAPopt and that blood pressures closer to MAPopt would be associated with less brain injury on MRI. We also tested a correlation-derived hemoglobin volume index (HVx) and single- and multi-window data processing methodology. Autoregulation was monitored in consecutive 3-h periods using near infrared spectroscopy in an observational study. The neonates had a mean MAP of 54 mmHg (standard deviation: 9) during hypothermia. Greater blood pressure above the MAPopt from single-window wHVx was associated with less injury in the paracentral gyri ( p = 0.044; n = 63), basal ganglia ( p = 0.015), thalamus ( p = 0.013), and brainstem ( p = 0.041) after adjustments for sex, vasopressor use, seizures, arterial carbon dioxide level, and a perinatal insult score. Blood pressure exceeding MAPopt from the multi-window, correlation HVx was associated with less injury in the brainstem ( p = 0.021) but not in other brain regions. We conclude that applying wavelet methodology to short autoregulation monitoring periods may improve the identification of MAPopt values that are associated with brain injury. Having blood pressure above MAPopt with an upper MAP of ~50-60 mmHg may reduce the risk of brain injury during therapeutic hypothermia. Though a cause-and-effect relationship cannot be inferred, the data support the need for randomized studies of autoregulation and brain injury in neonates with HIE., Competing Interests: JL, FN, and RC-V received research support from Medtronic for a separate study. JL was previously a paid consultant for Medtronic and JL is currently a paid consultant for Edwards Life Sciences. These arrangements have been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. Medtronic and Edwards Life Sciences had no role in the design of the current study, collection or analysis of the data, interpretation of the results, manuscript writing, or our decision to submit this manuscript for publication. Some methods used to measure and monitor autoregulation as described in this manuscript were patented by The Johns Hopkins University, listing KB as a co-inventor. These patents are exclusively licensed to Medtronic Inc. and KB received a portion of the licensing fee. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liu, Tekes, Perin, Chen, Soares, Massaro, Govindan, Parkinson, Chavez-Valdez, Northington, Brady and Lee.)

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 GABA B receptor subunit levels were decreased after HI, but unrelated to later seizure susceptibility. In contrast, Hp GABA A receptor α1 subunit (GABA A Rα1) levels were increased after HI. Adjusting the number of PV + INs for their GABA A Rα1 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., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Cooling delays, temperature outside 33-34 °C, and blood pressure below the mean arterial blood pressure with optimal cerebral autoregulation (MAP OPT ) might diminish neuroprotection from therapeutic hypothermia in neonates with hypoxic-ischemic encephalopathy (HIE). We hypothesized that longer time to reach temperature <34 °C and having temperature outside 33-34 °C would be associated with worse autoregulation and greater brain injury., Methods: Neonates with HIE had rectal temperature and near-infrared spectroscopy autoregulation monitoring during hypothermia (n = 63) and rewarming (n = 58). All underwent brain MRI, and a subset received diffusion tensor imaging MRI before day 10 (n = 41)., Results: Most neonates reached <34 °C at 3-6 h of life. MAP OPT was identified in 54/63 (86%) during hypothermia and in 53/58 (91%) during rewarming. Cooling time was not related to blood pressure deviation from MAP OPT . Later cooling was associated with lower ADC scalar in unilateral posterior centrum semiovale but not in other regions. Temperatures >34 °C were associated with blood pressure above MAP OPT but not with brain injury., Conclusions: In neonates who were predominantly cooled after 3 h, cooling time was not associated with autoregulation or overall brain injury. Blood pressure deviation above MAP OPT was associated with temperature >34 °C. Additional studies are needed in a more heterogeneous population., Impact: Cooling time to reach target hypothermia temperature within 6 h of birth did not affect cerebral autoregulation measured by NIRS in neonates with hypoxic-ischemic encephalopathy (HIE). Temperature fluctuations >33-34 °C were associated with blood pressures that exceeded the range of optimal autoregulatory vasoreactivity. Cooling time within 6 h of birth and temperatures >33-34 °C were not associated with qualitative brain injury on MRI. Regional apparent diffusion coefficient scalars on diffusion tensor imaging MRI were not appreciably affected by cooling time or temperature >33-34 °C. Additional research in a larger and more heterogeneous population is needed to determine how delayed cooling and temperatures beyond the target hypothermia range affect autoregulation and brain injury.

Background: Neonatal hypoxic-ischemic encephalopathy (HIE) is associated with dysfunctional cerebral autoregulation. Resistive index (RI) measured in the anterior cerebral artery on transfontanellar head ultrasound is a noninvasive measure of blood flow and may indicate autoregulation dysfunction. We tested whether RI was associated with brain injury on diffusion tensor imaging magnetic resonance imaging (MRI)., Materials and Methods: Seventy-five neonates who underwent therapeutic hypothermia for HIE were enrolled. Resistive index values were obtained from head ultrasound performed at the end of therapeutic hypothermia. Apparent diffusion coefficient scalars were measured on MRIs performed before day of life 10., Results: Lower RI was associated with lower apparent diffusion coefficient in the centrum semiovale, basal ganglia, thalamus, and posterior limb of the internal capsule. Combining RI and Apgar scores improved the ability to distinguish injury severity on MRI relative to either metric alone., Conclusions: Low RI correlated with worse brain injury on diffusion tensor imaging and may serve as an early marker of brain injury in cooled HIE neonates.

Neonatal hypoxic-ischemic encephalopathy (HIE) remains a common problem world-wide for infants born at term. The impact of HIE on long-term outcomes, especially into adulthood, is not well-described. To facilitate identification of biobehavioral biomarkers utilizing a translational platform, we sought to investigate the impact of HIE on executive function and cognitive outcomes into adulthood utilizing a murine model of HIE. HIE mice (unilateral common carotid artery occlusion to induce ischemia, followed by hypoxia with a FiO 2 of 0.08 for 45 min) and control mice were tested on discrimination and reversal touchscreen tasks (using their noses) shown to be sensitive to loss of basal ganglia or cortical function, respectively. We hypothesized that the HIE injury would result in deficits in reversal learning, revealing complex cognitive and executive functioning impairments. Following HIE, mice had a mild discrimination impairment as measured by incorrect responses but were able to learn the paradigm to similar levels as controls. During reversal, HIE mice required significantly more total trials, errors and correction trials across the paradigm. Analysis of specific stages showed that reversal impairments in HIE were driven by significant increases in all measured parameters during the late learning, striatal-mediated portion of the task. Together, these results support the concept that HIE occurring during the neonatal period results in abnormal neurodevelopment that persists into adulthood, which can impact efficient associated learning. Further, these data show that utilization of an established model of HIE coupled with touchscreen learning provides valuable information for screening therapeutic interventions that could mitigate these deficits to improve the long-term outcomes of this vulnerable population., (Copyright © 2020 Maxwell, Zimmerman, Pavlik, Newville, Carlin, Robinson, Brigman, Northington and Jantzie.)

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., Competing Interests: Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2019 by the American Society of Neuroimaging.)

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.

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 T 2 -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 f ADC) 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 f ADC 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.

There is increasing interest in applying physiological MRI in neonates, based on the premise that physiological parameters may provide an early biomarker of neonatal brain health and injury. Two commonly used techniques are oxygen extraction fraction (OEF) measurement using T 2 -relaxation-under-spin-tagging (TRUST) MRI and cerebral blood flow measurement using phase-contrast (PC) quantitative flow MRI, which collectively provide an assessment of the brain's oxygen consumption. However, prior research has only demonstrated proof of principle of these methods in neonates, without characterization or benchmarking of the techniques. This is because available time is limited in neonatal subjects, especially when scans are performed as add-ons to clinical scans (typically less than 5 min). The work presented aims to examine the TRUST and PC MRI sequences systematically in normal neonates, through research-dedicated scan sessions. A series of characterization and optimization studies were conducted in a total of 26 radiographically normal neonates on 3 T systems. Our results show that TRUST MRI at the superior sagittal sinus (SSS) provides an OEF measurement equivalent to that at the internal jugular vein (r = 0.80, n = 10), yet with shorter scan time. Lower resolution provided better precision in the TRUST measurement (p = 0.001, n = 9). Therefore, the preferred OEF measurement is to apply TRUST MRI at the SSS using a spatial resolution of 2.5 mm. For PC MRI, our results showed that non-gated PC MRI yielded blood flow measurements comparable to those from the more time-consuming gated approach in neonates (r = 0.89, n = 7). It was also found that blood flow could be overestimated by 18% when imaging resolution is larger than 0.3 mm (n = 7). Therefore, non-gated PC MRI with a spatial resolution of 0.3 mm is recommended for neonatal applications. In conclusion, this study verifies consistency of neonatal brain oxygenation and flow measurements across acquisition schemes and points to optimal strategies in parameter selection when using these sequences., (© 2019 John Wiley & Sons, Ltd.)

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., (Copyright © 2019. Published by Elsevier Ltd.)

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., (© 2019 S. Karger AG, Basel.)

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 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 < .001). Hippocampal DTI scalars were not associated with short-term outcomes., Conclusions: Quantitative DTI analysis of the hippocampus in neonates following HIE is a feasible technique to examine neuronal injury. Although DTI scalars were useful in identifying thalamic injury in our cohort, hippocampal DTI analysis did not provide additional information regarding hippocampal injury following HIE., (© 2018 by the American Society of Neuroimaging.)

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., (© 2019 S. Karger AG, Basel.)

Posthemorrhagic hydrocephalus of prematurity (PHHP) remains a global challenge. Early preterm infants (<32 weeks gestation), particularly those exposed to chorioamnionitis (CAM), are prone to intraventricular hemorrhage (IVH) and PHHP. We established an age-appropriate, preclinical model of PHHP with progressive macrocephaly and ventriculomegaly to test whether non-surgical neonatal treatment could modulate PHHP. We combined prenatal CAM and postnatal day 1 (P1, equivalent to 30 weeks human gestation) IVH in rats, and administered systemic erythropoietin (EPO) plus melatonin (MLT), or vehicle, from P2 to P10. CAM-IVH rats developed progressive macrocephaly through P21. Macrocephaly was accompanied by ventriculomegaly at P5 (histology), and P21 ( ex vivo MRI). CAM-IVH rats showed impaired performance of cliff aversion, a neonatal neurodevelopmental test. Neonatal EPO+MLT treatment prevented macrocephaly and cliff aversion impairment, and significantly reduced ventriculomegaly. EPO+MLT treatment prevented matted or missing ependymal motile cilia observed in vehicle-treated CAM-IVH rats. EPO+MLT treatment also normalized ependymal yes-associated protein (YAP) mRNA levels, and reduced ependymal GFAP-immunolabeling. Vehicle-treated CAM-IVH rats exhibited loss of microstructural integrity on diffusion tensor imaging, which was normalized in EPO+MLT-treated CAM-IVH rats. In summary, combined prenatal systemic inflammation plus early postnatal IVH caused progressive macrocephaly, ventriculomegaly and delayed development of cliff aversion reminiscent of PHHP. Neonatal systemic EPO+MLT treatment prevented multiple hallmarks of PHHP, consistent with a clinically viable, non-surgical treatment strategy.

Delayed hippocampal injury and memory impairments follow neonatal hypoxia-ischemia (HI) despite the use of therapeutic hypothermia (TH). Death of hippocampal pyramidal cells occurs acutely after HI, but characterization of delayed cell death and injury of interneurons (INs) is unknown. We hypothesize that injury of INs after HI is: (i) asynchronous to that of pyramidal cells, (ii) independent of injury severity, and (iii) unresponsive to TH. HI was induced in C57BL6 mice at p10 with unilateral right carotid ligation and 45 min of hypoxia (FiO 2  = 0.08). Mice were randomized to normothermia (36 °C, NT) or TH (31 °C) for 4 hr after HI and anesthesia-exposed shams were use as controls. Brains were studied at 24 hr (p11) or 8 days (p18) after HI. Vglut1, GAD65/67, PSD95, parvalbumin (PV) and calbindin-1 (Calb1) were measured. Cell death was assessed using cresyl violet staining and TUNEL assay. Hippocampal atrophy and astroglyosis at p18 were used to assess injury severity and to correlate with number of PV + INs. VGlut1 level decreased by 30% at 24 hr after HI, while GAD65/67 level decreased by ∼50% in forebrain 8 days after HI, a decrease localized in CA1 and CA3. PSD95 levels decreased in forebrain by 65% at 24 hr after HI and remained low 8 days after HI. PV + INs increased in numbers (per mm 2 ) and branching between p11 and p18 in sham mice but not in NT and TH mice, resulting in 21-52% fewer PV + INs in injured mice at p18. Calb1 protein and mRNA were also reduced in HI injured mice at p18. At p18, somatodendritic attrition of INs was evident in all injured mice without evidence of cell death. Neither hippocampal atrophy nor astroglyosis correlated with the number of PV + INs at p18. Thus, HI exposure has long lasting effects in the hippocampus impairing the development of the GABAergic system with only partial protection by TH independent of the degree of hippocampal injury. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Recent advances in diffusion MRI employ multiple diffusion encoding schemes with varying diffusion direction, weighting, and diffusion time to investigate specific microstructural properties in biological tissues. In this study, we examined time-dependent diffusion kurtosis contrast in adult mouse brains and in neonatal mouse brains after hypoxic-ischemic (HI) injury. In vivo diffusion kurtosis maps were acquired with a short diffusion time using an oscillating gradient spin echo (OGSE) sequence at 100 Hz and with a relatively long diffusion time (20 ms) using a pulsed gradient spin echo (PGSE) sequence. In the adult mouse brain, we found that the cortex and hippocampus showed larger differences between OGSE kurtosis and PGSE kurtosis than major white matter tracts. In neonatal mouse brains with unilateral HI injury, the OGSE kurtosis map overall provided stronger edema contrast than the PGSE kurtosis map, and the differences between OGSE and PGSE kurtosis measurements in the edema region reflected heterogeneity of injury. This is the first in vivo study that has demonstrated multi-direction OGSE kurtosis contrasts in the mouse brain. Comparing PGSE and OGSE kurtosis measures may provide additional information on microstructural changes after ischemic stroke., (Copyright © 2018 John Wiley & Sons, Ltd.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cerebral palsy (CP) is the leading cause of motor impairment for children worldwide and results from perinatal brain injury (PBI). To test novel therapeutics to mitigate deficits from PBI, we developed a rat model of extreme preterm birth (<28 weeks of gestation) that mimics dual intrauterine injury from placental underperfusion and chorioamnionitis. We hypothesized that a sustained postnatal treatment regimen that combines the endogenous neuroreparative agents erythropoietin (EPO) and melatonin (MLT) would mitigate molecular, sensorimotor, and cognitive abnormalities in adults rats following prenatal injury. On embryonic day 18 (E18), a laparotomy was performed in pregnant Sprague-Dawley rats. Uterine artery occlusion was performed for 60 min to induce placental insufficiency via transient systemic hypoxia-ischemia, followed by intra-amniotic injections of lipopolysaccharide, and laparotomy closure. On postnatal day 1 (P1), approximately equivalent to 30 weeks of gestation, injured rats were randomized to an extended EPO + MLT treatment regimen, or vehicle (sterile saline) from P1 to P10. Behavioral assays were performed along an extended developmental time course ( n  = 6-29). Open field testing shows injured rats exhibit hypermobility and disinhibition and that combined neonatal EPO + MLT treatment repairs disinhibition in injured rats, while EPO alone does not. Furthermore, EPO + MLT normalizes hindlimb deficits, including reduced paw area and paw pressure at peak stance, and elevated percent shared stance after prenatal injury. Injured rats had fewer social interactions than shams, and EPO + MLT normalized social drive. Touchscreen operant chamber testing of visual discrimination and reversal shows that EPO + MLT at least partially normalizes theses complex cognitive tasks. Together, these data indicate EPO + MLT can potentially repair multiple sensorimotor, cognitive, and behavioral realms following PBI, using highly translatable and sophisticated developmental testing platforms.

Purpose of Review: The rapid progress in biomarker science is on the threshold of significantly changing clinical care for infants in the neonatal ICU. 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; Specific nonbrain markers of cardiac health and global asphyxia continue to provide information on brain injury after hypoxic-ischemic encephalopathy (HIE). Prediction of injury in the piglet hypoxia-ischemia model is improved with the use of a combination score of plasma metabolites. In a neonatal piglet model of perinatal hypoxia-ischemia, a systemic proinflammatory surge of cytokines has been identified after rewarming from therapeutic hypothermia. New biomarkers identified recently include osteopontin, activin A, neutrophil gelatinase-associated lipocalin, secretoneurin, Tau and neurofilament light protein. 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.