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.

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.

epilepsy. Higher levels of pro-inflammatory cytokines IL-6 and TNF-α within the IL-1β pathway were significantly associated with epilepsy.

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.

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.

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 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 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]

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.

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

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.

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 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.

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 (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 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).

Neonatal cytokine levels in children with acute symptomatic/early seizures (n = 151) compared to those without seizures (n = 11). Levels are plotted from 0 to 200 pg/mL, except for

interleukin (IL)-12 and IL-13, which are plotted from 0 to 60 pg/mL. *p