Early changes in pro-inflammatory cytokine levels in neonates with encephalopathy are associated with remote epilepsy

ABSTRACT BACKGROUND Neonatal seizures are associated with adverse neurologic sequelae including epilepsy in childhood. Here we aim to determine whether levels of cytokines in neonates with

brain injury are associated with acute symptomatic seizures or remote epilepsy. METHODS This is a cohort study of term newborns with encephalopathy at UCSF between 10/1993 and 1/2000 who had

dried blood spots. Maternal, perinatal/postnatal, neuroimaging, and epilepsy variables were abstracted by chart review. Logistic regression was used to compare levels of cytokines with

acute seizures and the development of epilepsy. RESULTS In a cohort of 26 newborns with neonatal encephalopathy at risk for hypoxic ischemic encephalopathy with blood spots for analysis,

diffuse alterations in both pro- and anti-inflammatory cytokine levels were observed between those with (11/28, 39%) and without acute symptomatic seizures. Seventeen of the 26 (63%)

patients had >2 years of follow-up and 4/17 (24%) developed epilepsy. Higher levels of pro-inflammatory cytokines IL-6 and TNF-α within the IL-1β pathway were significantly associated

with epilepsy. CONCLUSIONS Elevations in pro-inflammatory cytokines in the IL-1β pathway were associated with later onset of epilepsy. Larger cohort studies are needed to confirm the

predictive value of these circulating biomarkers. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS ELEVATED SERUM IL-10 IS

ASSOCIATED WITH SEVERITY OF NEONATAL ENCEPHALOPATHY AND ADVERSE EARLY CHILDHOOD OUTCOMES Article Open access 05 March 2021 THE ASSOCIATION BETWEEN EARLY-ONSET SEPSIS AND NEONATAL

ENCEPHALOPATHY Article 10 January 2022 CSF NEOPTERIN AND BETA-2-MICROGLOBULIN AS INFLAMMATION BIOMARKERS IN NEWBORNS WITH HYPOXIC–ISCHEMIC ENCEPHALOPATHY Article 06 April 2022 INTRODUCTION

Neonatal seizures are an indicator of neurologic dysfunction with an incidence of 2.8–4.4/1000 live births.1,2 Neonates with seizures are at high risk for a range of adverse neurologic

sequelae compared to those without seizures, with up to 25% developing remote epilepsy.3,4 Risk factors for epilepsy include severity of encephalopathy, severity and type of brain injury,

abnormal electroencephalogram (EEG) background, and seizure frequency.3,4,5 Together these variables can identify a high-risk group with an approximately 50% chance of developing

epilepsy.3,4,5 Additional predictors of epilepsy, however, are needed in order to improve stratification, to better inform families, and to guide therapeutic studies that can alter epilepsy

outcomes.6,7 Alterations in the levels of inflammatory cytokines, in particular the IL-1β pathway, may serve as biomarkers of neurologic disease. These molecules are secreted by activated

neuroglia often within an hour of an inciting central nervous system (CNS) insult, including status epilepticus, stroke, and infection.8,9,10 IL-1β activates its endogenous receptor with

resultant increases in neuronal excitability.11,12,13 After an initial CNS insult, ongoing inflammation may alter neuronal plasticity with network reorganization through several

transcriptionally regulated effects, with potential for aberrant and epileptogenic circuits.14,15,16,17,18 Activation of the pathway enhances the permeability of an already dysfunctional

blood–brain barrier, allowing for movement and detection of these proteins into the peripheral circulation, raising the possibility of their utility as a biomarker of disease.19,20 In this

study, we aimed to determine whether the levels of cytokines in neonates with brain injury are associated with acute symptomatic seizures and the development of epilepsy in childhood. We

hypothesize that changes in a diffuse set of neonatal cytokines will be associated with acute seizures, though only increases in cytokines within the pro-inflammatory IL-1β pathway will be

associated with remote epilepsy. METHODS SUBJECTS This is a nested cohort study within a longitudinal investigation of term and near-term newborns at risk of neonatal encephalopathy.21,22 As

previously reported, newborns were recruited who were admitted to the Intensive Care Nursery at UCSF and a nearby county hospital between 10/1993 and 1/2000 and had any of the following:

(1) umbilical artery pH <7.1, (2) umbilical artery base deficit >10, (3) Apgar score ≤5 at 5 min of life, or (4) overt neonatal encephalopathy as assessed by a neonatologist. This

cohort was assembled before the adoption of therapeutic hypothermia. Neonates were excluded if there was evidence of major congenital malformations, congenital metabolic diseases, or

perinatal or intrauterine infection. The original cohort enrolled 125 neonates, 62 of which had cytokine levels evaluated from dried blood spots. Here we aimed to evaluate cytokine levels in

term neonates with high risk of brain injury. We applied additional exclusion criteria to the study base, excluding neonates <37 weeks gestational age, without both clinical and

physiologic evidence of hypoxia–ischemia (and therefore not meeting our institutional therapeutic hypothermia criteria),23 deceased during the birth admission, and dried blood spots

collected < 24 or >120 h after birth to allow for evaluation of relevant cytokines at peak levels after an acute neurologic injury.21,24,25 Epilepsy classification was restricted to

those with at least 2 years of follow-up to allow for the development of epilepsy.3,4] MEASUREMENTS CYTOKINES Cytokine levels were previously evaluated, and levels were reported in an

investigation evaluating their association with magnetic resonance spectroscopy (MRS) and development.21 In brief, dried blood spots were obtained from heel-stick blood as a part of

California’s newborn screening program. Dried blood spots were analyzed by recycling immunoaffinity chromatography.26 Twenty-five-microliter samples were injected into serially connected

microcolumns, each containing a different immobilized capture antibody interleukin (IL)-1, IL-6, IL-8, IL-9, IL-12, IL-13, and tumor necrosis factor (TNF)-α).22 Analytes were released by

treatment with acidic buffer and measured by laser-induced fluorescence. CLINICAL DATA Trained research assistants prospectively abstracted demographics and birth delivery data.21

Encephalopathy scores were determined by expert review.21,22 Magnetic resonance imaging (MRI) injury scores were determined by combining scores for abnormalities in the deep gray nuclei and

white matter.27 Two authors (A.L.N., H.C.G.) reviewed charts for encephalopathy etiology, presence of clinical and/or electrographic neonatal acute symptomatic seizures, and epilepsy as

defined by the International League Against Epilepsy 2014 criteria.28 DATA ANALYSIS Analyses were performed using the Stata 15.1 software (Stata Corp, College Station, TX). Chi-square test

was used to compare categorical variables, Whitney Rank-Sum test was used to compare ordinal variables, and _t_ test was used to compare continuous variables with acute seizures or epilepsy.

For the analyses of cytokines, linear regression was used to compare levels with continuous variables, Kruskal–Wallis test was used with ordinal variables, and t tests or analysis of

variance were used for comparisons of categorical predictors and outcome variables. We performed validation using sensitivity analyses with nonparametric tests. The area under the receiver

operator curve (AUC) for cytokine levels and epilepsy were estimated using non-parametric methods. Rv3.2.0 (R Foundation for Statistical Computing, Vienna, Austria) was used for clustering

methods, using the gplots package. For each patient, cytokine values were scaled by subtracting the mean cytokine value across all patients from each particular patient cytokine level. This

value was then divided by the standard deviation of the particular cytokine. Hierarchical agglomerative clustering algorithms were used to evaluate clusters of patients within the plasma

cytokine determinants. Data were standardized as _Z_-scores by subtracting mean and divided by standard deviation for each cytokine. Pearson’s correlation similarity metric and Ward linkage

function were used for hierarchical agglomerative cluster analysis. For each clustering algorithm, dendrograms, heatmaps, and bubblemaps were created to visualize the clusters and scaled

cytokine values. Dark blue colors on the heatmaps represent low cytokine values, white colors represent average values, and red represent high values. Larger bubbles on bubblemaps correspond

to higher cytokine values. The protocol was approved by the Committee for Human Research at the University of California, San Francisco and voluntary informed consent was obtained from

parents or legal guardians. RESULTS COHORT CHARACTERISTICS From a study base of 62 neonates with encephalopathy, we excluded 7 (11%) neonates < 37 weeks gestational age, 10 (16%) infants

without clinical and physiologic evidence of hypoxia–ischemia, 2 (3%) deceased during the birth admission, and 10 (16%) infants with dried blood spots collected <24 or >120 h after

birth. Seven (11%) of the 62 infants had missing clinical details on birth history and follow-up, leaving 26 infants for evaluation of acute symptomatic seizures. Among these 26 infants, 22

(85%) had radiologic evidence consistent with hypoxia–ischemia, 3 (12%) had radiologic evidence of ischemic or hemorrhagic stroke, and 1 (4%) had laboratory-confirmed meningitis. ACUTE

SEIZURES Fifteen (58%) of the 26 neonates had acute symptomatic seizures. Seizure onset was within 48 h of life in all subjects. Five neonates had rare (<7) seizures, 2 had frequent

recurrent seizures (≥7), and 2 had status epilepticus. The remaining 6 had greater than 1 seizure, but the precise burden could not be determined by chart review. Neonates with acute

seizures had higher encephalopathy scores and a trend for higher MRI injury scores compared to those without acute seizures (Table 1). The groups did not differ on other demographic,

clinical, and maternal risk factors. REMOTE EPILEPSY Seventeen (65%) of 26 neonates had >2 years of follow-up data for review to evaluate epilepsy onset after the neonatal period. Four of

the 17 (24%) developed epilepsy, all of whom had a history of acute symptomatic seizures. There were no significant differences in demographic, clinical, and maternal risk factors between

those with and without remote epilepsy (Table 1). CYTOKINE ANALYSES Cytokine levels were evaluated at 2.5 days of life (95% confidence interval: 2.1–2.9). There were no significant

differences in timing of DBS collection between those with and without acute symptomatic seizure or between those with and without remote epilepsy. Cytokine levels did not vary by sex, race,

gestational age, maternal age, encephalopathy etiology, encephalopathy score, or MRI severity score. Neonates with acute symptomatic seizures had broad difference in cytokines levels in

comparison to those without acute seizures (Fig. 1). Those with acute seizures had higher levels of IL-1, -6, -8, -9, -13, and TNF-α, as well as lower levels of IL-12, compared to those

without acute seizures. In contrast, neonates who developed epilepsy had higher levels of cytokines specific to the IL-1β pathway, including IL-6 and TNF-α, as well as IL-9 (Fig. 2). There

was trend for higher IL-1 levels (combined α and β subunits) in those who developed epilepsy (_p_ = 0.07). We evaluated whether cytokine levels can discriminate between those with and

without remote epilepsy with the receiver operator curve characteristics. The AUC for cytokines within the pro-inflammatory IL-1β pathway ranged from 0.87 to 0.88, demonstrating a strong

effect and superior performance to IL-12 and IL-13 (Table 2). Hierarchical agglomerative clustering algorithms revealed difference in patterns of cytokine level changes in neonates with and

without later onset of epilepsy (Fig. 3). Dendrograms (Fig. 3) and bubblemaps/heatmaps (Fig. 4) confirmed correlations of cytokines within the pro-inflammatory IL-1β pathway (IL-1, IL-6,

IL-8, TNF-α). IL-12 and IL-13, which are involved in disparate signaling pathways, were not correlated with the other cytokines measured. DISCUSSION Here we evaluated the association of

inflammatory cytokines with acute symptomatic seizures and development of remote epilepsy in a cohort of neonates with encephalopathy. Acute symptomatic seizures in neonates with

encephalopathy were associated with broad changes in cytokines in both pro- and anti-inflammatory cytokine levels. However, only increased levels of pro-inflammatory cytokines in the IL-1β

pathway, and to a lesser extent, IL-9 were associated with later development of epilepsy. This group previously reported that, in this cohort of neonates with encephalopathy, elevations in

IL-1, IL-6, and IL-8 on dried blood spots were correlated with elevated lactate peaks on MRS.21 Similarly, “The Extremely Low Gestational Age Newborn Study” (ELGAN) study evaluated several

cytokines in premature infants born <28 weeks gestation using analyses of dried blood spots.25 Changes in cytokine levels in several pro- and anti-inflammatory pathways were associated

with the presence of intraventricular hemorrhage and/or white matter injury.29 As neonatal brain injury is a risk factor for the development of acute symptomatic seizures,3,4 it is not

surprising that we found acute symptomatic seizures after neonatal brain injury were associated with alterations in a number of pro- and anti-inflammatory cytokine pathways. Our findings

corroborate data from a cohort of 13 neonates with hypoxic–ischemic encephalopathy, where serum levels of IL-1 receptor antagonist (IL-1RA; an inhibitor of the IL-1 pathway) were lower and

levels of IL-8 were higher among neonates with seizures compared to controls,24 though, in all clinical studies evaluating cytokines levels in neonates to date, the development of remote

epilepsy was not assessed. Our finding that _neonatal_ elevations of cytokines within the IL-1β pathway are associated with the development of epilepsy in childhood is novel. This pathway,

however, has been associated with epilepsy in several other clinical populations, including adults with traumatic brain injury and post-traumatic epilepsy.30,31 Therefore, this pathway may

serve as a marker of epilepsy across several types of brain injury with the potential for understanding mechanisms of epileptogenesis.32 Cytokines in the IL-1β pathway modulate the

excitatory NMDA receptor and the inhibitory GABA receptor on neurons and astrocytes, resulting in lower seizure thresholds, which can lead to a positive feedback loop with perpetuation of

seizure activity and brain inflammation.11,12,13,33,34 After an initial brain injury, this ongoing inflammation can alter neuronal plasticity with network reorganization through several

transcriptionally regulated effects, with potential for aberrant and epileptogenic circuits.14,15,16,17,18 In addition to differences in the IL-1β pathway, we also found that increased

levels of IL-9 were associated with later development of epilepsy. The role of IL-9 in neurologic injury is less understood, with an emerging role in encephalomyelitis and multiple

sclerosis.35 Further investigations are needed to validate this finding. Limitations of our study include our small sample size, which did not allow us to evaluate for potential confounding

by brain injury severity on cytokine levels and later development of epilepsy. The etiology of neonatal encephalopathy, however, was similar to that observed in larger cohorts of term

neonates. While the prevalence of acute symptomatic seizures in neonates with encephalopathy is higher than that of contemporary cohorts (58%), it is consistent with the prevalence of acute

symptomatic seizures in neonates before therapeutic hypothermia was adopted.36 In this cohort, the evaluation of acute symptomatic seizures was not standardized. During the time period in

which the cohort was assembled, EEG was not consistently used to evaluate for subclinical seizures in the neonatal period. While this could result in misclassification of neonates without

“acute symptomatic seizure,” this would reduce our ability to detect an association of cytokine levels with seizures. The prevalence of epilepsy (24%) in this cohort is similar to that

observed in larger cohort studies.37 Owing to the retrospective cohort design, several patients were lost to follow-up for evaluation of epilepsy. While this could introduce bias, we

anticipate that cases of epilepsy were unlikely to be misclassified owing to referral patterns within our catchment area. Here we used dried blood spots to measure cytokine levels. This

sampling results in increased measurement variability compared to measurement of cytokine levels in serum or plasma likely owing to the use of capillary blood, sample spread, and activity of

leukocytes during sample storage without early centrifugation.38,39 We would anticipate the use of serum or plasma would decrease the spread of cytokine levels by group and therefore

strengthen the observed associations. Here measurement of IL-1 levels included both the IL-1α and β subunits. The IL-1β subunit is responsible for inflammation after a neurologic injury,

whereas the α subunit is largely skin derived and would not be expected to change after brain injury.40 Levels of IL-1α and IL-1β are similar in circulation; therefore inclusion of IL-1α in

measurements would dilute the association of IL-1β with our outcomes. If subunit composition analysis of IL-1β were possible, we hypothesize that the trend for increased IL-1 levels

associated with epilepsy would have a larger effect size and become statistically significant. Our findings support the utility of circulating cytokines as a predictor of epilepsy after

neonatal brain injury. Larger, prospective studies in contemporary cohorts of neonates are necessary to validate these findings and evaluate for interactions between changes in cytokines

levels and the IL-1β pathway with clinical factors known to increase risk of remote epilepsy such as brain injury and acute seizure severity. REFERENCES * Ronen, G. M., Penney, S. &

Andrews, W. The epidemiology of clinical neonatal seizures in Newfoundland: a population-based study. _J. Pediatr._ 134, 71–75 (1999). Article  CAS  Google Scholar  * Lanska, M. J., Lanska,

D. J., Baumann, R. J. & Kryscio, R. J. A population-based study of neonatal seizures in Fayette County, Kentucky. _Neurology_ 45, 724–732 (1995). Article  CAS  Google Scholar  * Fox, C.

K., Glass, H. C., Sidney, S., Smith, S. E. & Fullerton, H. J. Neonatal seizures triple the risk of a remote seizure after perinatal ischemic stroke. _Neurology_ 86, 2179–2186 (2016).

Article  Google Scholar  * Glass, H. C. et al. Risk factors for epilepsy in children with neonatal encephalopathy. _Pediatr. Res._ 70, 535–540 (2011). Article  Google Scholar  * Glass, H.

C., Numis, A. L., Gano, D., Bali, V. & Rogers, E. E. Outcomes after acute symptomatic seizures in children admitted to a neonatal neurocritical care service. _Pediatr. Neurol._ 84, 39–45

(2018). Article  Google Scholar  * Sillanpaa, M., Camfield, P. & Camfield, C. Predicting long-term outcome of childhood epilepsy in Nova Scotia, Canada, and Turku, Finland. Validation

of a simple scoring system. _Arch. Neurol._ 52, 589–592 (1995). Article  CAS  Google Scholar  * Ronen, G. M., Buckley, D., Penney, S. & Streiner, D. L. Long-term prognosis in children

with neonatal seizures: a population-based study. _Neurology_ 69, 1816–1822 (2007). Article  Google Scholar  * Vezzani, A., Friedman, A. & Dingledine, R. J. The role of inflammation in

epileptogenesis. _Neuropharmacology_ 69, 16–24 (2013). Article  CAS  Google Scholar  * De Simoni, M. G. et al. Inflammatory cytokines and related genes are induced in the rat hippocampus by

limbic status epilepticus. _Eur. J. Neurosci._ 12, 2623–2633 (2000). Article  Google Scholar  * Rooker, S. et al. Spatiotemporal pattern of neuroinflammation after impact-acceleration closed

head injury in the rat. _Mediat. Inflamm._ 2006, 90123 (2006). Article  Google Scholar  * Wang, S., Cheng, Q., Malik, S. & Yang, J. Interleukin-1beta inhibits gamma-aminobutyric acid

type A (GABA(A)) receptor current in cultured hippocampal neurons. _J. Pharm. Exp. Ther._ 292, 497–504 (2000). CAS  Google Scholar  * Lai, A. Y., Swayze, R. D., El-Husseini, A. & Song,

C. Interleukin-1 beta modulates AMPA receptor expression and phosphorylation in hippocampal neurons. _J. Neuroimmunol._ 175, 97–106 (2006). Article  CAS  Google Scholar  * Roseti, C. et al.

GABAA currents are decreased by IL-1beta in epileptogenic tissue of patients with temporal lobe epilepsy: implications for ictogenesis. _Neurobiol. Dis._ 82, 311–320 (2015). Article  CAS 

Google Scholar  * Pugazhenthi, S., Zhang, Y., Bouchard, R. & Mahaffey, G. Induction of an inflammatory loop by interleukin-1beta and tumor necrosis factor-alpha involves NF-kB and STAT-1

in differentiated human neuroprogenitor cells. _PLoS ONE_ 8, e69585 (2013). Article  CAS  Google Scholar  * Ross, F. M., Allan, S. M., Rothwell, N. J. & Verkhratsky, A. A dual role for

interleukin-1 in LTP in mouse hippocampal slices. _J. Neuroimmunol._ 144, 61–67 (2003). Article  CAS  Google Scholar  * del Rey, A., Balschun, D., Wetzel, W., Randolf, A. & Besedovsky,

H. O. A cytokine network involving brain-borne IL-1beta, IL-1ra, IL-18, IL-6, and TNFalpha operates during long-term potentiation and learning. _Brain Behav. Immun._ 33, 15–23 (2013).

Article  Google Scholar  * Yin, P. et al. Neonatal immune challenge exacerbates seizure-induced hippocampus-dependent memory impairment in adult rats. _Epilepsy Behav._ 27, 9–17 (2013).

Article  Google Scholar  * Balosso, S. et al. A novel non-transcriptional pathway mediates the proconvulsive effects of interleukin-1beta. _Brain_ 131, 3256–3265 (2008). Article  Google

Scholar  * Qin, L. J., Gu, Y. T., Zhang, H. & Xue, Y. X. Bradykinin-induced blood-tumor barrier opening is mediated by tumor necrosis factor-alpha. _Neurosci. Lett._ 450, 172–175 (2009).

Article  CAS  Google Scholar  * Didier, N. et al. Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular

endothelial cell permeability. _J. Neurochem._ 86, 246–254 (2003). Article  CAS  Google Scholar  * Bartha, A. I. et al. Neonatal encephalopathy: association of cytokines with MR spectroscopy

and outcome. _Pediatr. Res._ 56, 960–966 (2004). Article  CAS  Google Scholar  * Foster-Barber, A. & Ferriero, D. M. Neonatal encephalopathy in the term infant: neuroimaging and

inflammatory cytokines. _Ment. Retard. Dev. Disabil. Res. Rev._ 8, 20–24 (2002). Article  Google Scholar  * Bonifacio, S. L. et al. Perinatal events and early magnetic resonance imaging in

therapeutic hypothermia. _J. Pediatr._ 158, 360–365 (2011). Article  Google Scholar  * Youn, Y. A. et al. Serial examination of serum IL-8, IL-10 and IL-1Ra levels is significant in neonatal

seizures induced by hypoxic-ischaemic encephalopathy. _Scand. J. Immunol._ 76, 286–293 (2012). Article  CAS  Google Scholar  * O’Shea, T. M. et al. Elevated concentrations of

inflammation-related proteins in postnatal blood predict severe developmental delay at 2 years of age in extremely preterm infants. _J. Pediatr._ 160, 395.e4–401.e4 (2012). Article  Google

Scholar  * Phillips, T. M. & Krum, J. M. Recycling immunoaffinity chromatography for multiple analyte analysis in biological samples. _J. Chromatogr. B Biomed. Sci. Appl._ 715, 55–63

(1998). Article  CAS  Google Scholar  * Barkovich, A. J. et al. Prediction of neuromotor outcome in perinatal asphyxia: evaluation of MR scoring systems. _AJNR Am. J. Neuroradiol._ 19,

143–149 (1998). CAS  Google Scholar  * Hajnal, B. L., Sahebkar-Moghaddam, F., Barnwell, A. J., Barkovich, A. J. & Ferriero, D. M. Early prediction of neurologic outcome after perinatal

depression. _Pediatr. Neurol._ 21, 788–793 (1999). Article  CAS  Google Scholar  * Leviton, A. et al. Systemic inflammation, intraventricular hemorrhage, and white matter injury. _J. Child

Neurol._ 28, 1637–1645 (2013). Article  Google Scholar  * Diamond, M. L. et al. IL-1beta associations with posttraumatic epilepsy development: a genetics and biomarker cohort study.

_Epilepsia_ 55, 1109–1119 (2014). Article  CAS  Google Scholar  * Gallentine, W. B. et al. Plasma cytokines associated with febrile status epilepticus in children: a potential biomarker for

acute hippocampal injury. _Epilepsia_ 58, 1102–1111 (2017). Article  CAS  Google Scholar  * Vezzani, A., Aronica, E., Mazarati, A. & Pittman, Q. J. Epilepsy and brain inflammation. _Exp.

Neurol._ 244, 11–21 (2013). Article  CAS  Google Scholar  * Chiavegato, A., Zurolo, E., Losi, G., Aronica, E. & Carmignoto, G. The inflammatory molecules IL-1beta and HMGB1 can rapidly

enhance focal seizure generation in a brain slice model of temporal lobe epilepsy. _Front. Cell. Neurosci._ 8, 155 (2014). Article  Google Scholar  * Tao, A. F. et al. The pro-inflammatory

cytokine interleukin-1beta is a key regulatory factor for the postictal suppression in mice. _CNS Neurosci. Ther._ 21, 642–650 (2015). Article  CAS  Google Scholar  * Ding, X. et al. IL-9

signaling affects central nervous system resident cells during inflammatory stimuli. _Exp. Mol. Pathol._ 99, 570–574 (2015). Article  CAS  Google Scholar  * Orbach, S. A., Bonifacio, S. L.,

Kuzniewicz, M. W. & Glass, H. C. Lower incidence of seizure among neonates treated with therapeutic hypothermia. _J. Child Neurol._ 29, 1502–1507 (2014). Article  Google Scholar  *

Pisani, F., Facini, C., Pavlidis, E., Spagnoli, C. & Boylan, G. Epilepsy after neonatal seizures: literature review. _Eur. J. Paediatr. Neurol._ 19, 6–14 (2015). Article  Google Scholar

  * Skogstrand, K. et al. Effects of blood sample handling procedures on measurable inflammatory markers in plasma, serum and dried blood spot samples. _J. Immunol. Methods_ 336, 78–84

(2008). Article  CAS  Google Scholar  * Schiffer, J. M. et al. Quantitative assessment of anthrax vaccine immunogenicity using the dried blood spot matrix. _Biologicals_ 41, 98–103 (2013).

Article  Google Scholar  * Feldmeyer, L., Werner, S., French, L. E. & Beer, H. D. Interleukin-1, inflammasomes and the skin. _Eur. J. Cell Biol._ 89, 638–644 (2010). Article  CAS  Google

Scholar  Download references ACKNOWLEDGEMENTS The authors thank Dr. Agnes Bartha for providing the groundwork for this dataset This work was supported by the National Institutes of Health

Grants RR 01271, NS 35902, NS 40117, L40 NS094060, and the American Academy of Neurology Clinical Research Training Fellowship in Epilepsy. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Department of Neurology, UCSF, San Francisco, CA, USA Adam L. Numis, Audrey Foster-Barber, Donna M. Ferriero & Hannah C. Glass * Department of Pediatrics, UCSF, San Francisco, CA, USA

Adam L. Numis, Audrey Foster-Barber, Elizabeth E. Rogers, Donna M. Ferriero & Hannah C. Glass * Vitalant Research Institute, San Francisco, CA, USA Xutao Deng * Department of Radiology,

UCSF, San Francisco, CA, USA A. James Barkovich * Department of Epidemiology and Biostatistics, UCSF, San Francisco, CA, USA Hannah C. Glass Authors * Adam L. Numis View author publications

You can also search for this author inPubMed Google Scholar * Audrey Foster-Barber View author publications You can also search for this author inPubMed Google Scholar * Xutao Deng View

author publications You can also search for this author inPubMed Google Scholar * Elizabeth E. Rogers View author publications You can also search for this author inPubMed Google Scholar *

A. James Barkovich View author publications You can also search for this author inPubMed Google Scholar * Donna M. Ferriero View author publications You can also search for this author

inPubMed Google Scholar * Hannah C. Glass View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conception and design of the study: A.L.N.,

D.M.F., H.C.G. Acquisition and analysis of data: A.L.N., X.D., E.E.R., A.J.B., D.M.F., H.C.G. Drafting of manuscript: A.L.N., H.C.G. Critical revision and final approval of the version to be

published: all the authors. CORRESPONDING AUTHOR Correspondence to Adam L. Numis. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

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THIS ARTICLE CITE THIS ARTICLE Numis, A.L., Foster-Barber, A., Deng, X. _et al._ Early changes in pro-inflammatory cytokine levels in neonates with encephalopathy are associated with remote

epilepsy. _Pediatr Res_ 86, 616–621 (2019). https://doi.org/10.1038/s41390-019-0473-x Download citation * Received: 31 January 2019 * Revised: 05 June 2019 * Accepted: 14 June 2019 *

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