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. Author manuscript; available in PMC: 2023 May 1.
Published in final edited form as: Am J Perinatol. 2020 Oct 10;39(7):732–749. doi: 10.1055/s-0040-1717072

Blood Biomarkers and 6–7 Year Childhood Outcomes Following Neonatal Encephalopathy

Athina Pappas 1, Seetha Shankaran 1, Scott A McDonald 2, Waldemar A Carlo 3, Abbot R Laptook 4, Jon E Tyson 5, Abhik Das 6, Kristin Skogstrand 7, David M Hougaard 7, Rosemary D Higgins 8; Hypothermia Extended Follow-up Subcommittee of the Eunice Kennedy Shriver NICHD Neonatal Research Network
PMCID: PMC8765716  NIHMSID: NIHMS1768854  PMID: 33038899

Abstract

OBJECTIVE:

This study aimed to profile the cytokine/chemokine response from day 0–7 in infants (≥ 36 week GA) with neonatal encephalopathy and to explore the association with long-term outcomes.

STUDY DESIGN:

This was a secondary study of the NICHD Neonatal Research Network randomized controlled trial of whole body hypothermia for neonatal encephalopathy. Eligible infants with moderate-severe neonatal encephalopathy were randomized to cooling or normothermia. Blood spots were collected on days 0–1, 2–4 and 6–7. 24 cytokines/chemokines were measured using a multiplex platform. Surviving infants underwent neurodevelopmental assessment at 6–7 years. Primary outcome was death or moderate-severe impairment defined by any of: IQ<70, moderate-severe cerebral palsy, blindness, hearing impairment or epilepsy.

RESULTS:

Cytokine blood spots were collected from 109 participants. 99 /109 (91%) were assessed at 6–7 years; 54/99 (55%) developed death/impairment. Neonates who died or were impaired had lower early RANTES and higher day 7 MCP-1 levels than neonates who survived without impairment. Though TNFα levels had no association with death/impairment, higher day 0–1 levels were observed among neonates who died/developed CP. On multiple regression analysis adjusted for center, treatment group, sex, race and level of HIE, higher RANTES was inversely associated with death/impairment (odds ratio (OR) 0.31, 95% CI 0.13–0.74), while day 7 MCP-1 level was directly associated with death/impairment (odds ratio (OR) 3.70, 95% CI 1.42–9.61). Targeted cytokine/chemokine levels demonstrated little variation with hypothermia treatment.

CONCLUSIONS:

RANTES and MCP-1 levels in the first week of life may provide potential targets for future therapies among neonates with encephalopathy.

Keywords: Hypoxic-ischemic encephalopathy, Neuro-developmental Impairment, Blood biomarkers, Cytokines, Chemokines, RANTES, MCP-1

INTRODUCTION:

Cytokines and chemokines mediate brain injury in the developing brain in preclinical models and may be useful biomarkers of injury and disease progression in neonatal encephalopathy (NE) (1, 2). Studies of term infants with NE report associations between elevated interleukin-6 (3–5), interleukin-8 (4, 6), interleukin-1β (4) and lower interleukin-12 levels (4) and death or neurodevelopmental impairment in infancy and early childhood. A magnetic resonance spectroscopy study in term neonates with encephalopathy also reported a significant association between elevated cytokine levels (interleukin-1β, IL-6, IL-8 and tumor necrosis factor-α) and elevated lactate/choline by imaging in the deep gray nuclei of the brain (4). Recent studies report on plasma cytokines and neurodevelopmental outcomes among neonates treated with hypothermia for NE (7–9). Jenkins et al found that elevated IL-6 and MCP-1 levels within 9 hours after birth and low MIP1-α levels at 60–70 hours of age were associated with death or severe neurodevelopmental impairment at 12 months(7). Chalak et al reported that elevated GFAP, IL1, IL6, IL8, tumor necrosis factor-α and interferon-ɣ (IFN-ɣ) at 6–24 hours were associated with abnormal outcome at 15–18 months(8). Massaro et al reported that elevated plasma levels of IL1β, IL6, IL8, IL10, IL13, TNFα and IFNɣ were associated with increased severity of brain injury by MRI, but not one year outcome(9).

Dried blood spot samples are an alternative specimen collection medium and may permit investigators the ability to store samples over prolonged periods and then pool data sets. Pioneering work by Nelson et al utilized stored neonatal blood spot samples to assess inflammatory cytokines among children with and without subsequent cerebral palsy (CP)(10), often seen following NE. Concentrations of IL-1β, IL-8, IL-9, tumor necrosis factor-α (TNF-α) and Regulated upon Activation Normal T-Cell Expressed and Secreted (RANTES) were higher among children diagnosed with spastic diplegia, but not quadriplegia. Moreover, levels of macrophage inflammatory proteins (MIP-1α and 1β) and monocyte chemotactic proteins (MCP-1 and 2) were significantly higher among children with CP as compared to those without CP.

We conducted an exploratory study to profile the cytokine/chemokine response from dried blood spot samples (DBSS) collected from day 0–7 in ≥ 36 week GA infants with moderate-severe NE and to examine the association with 6–7 year neurodevelopmental outcomes. Outcomes at 6–7 years provide a more accurate appraisal of functional childhood outcomes following neonatal encephalopathy as compared with the very early evaluations reported in other studies. We hypothesized that inflammatory cytokines and chemokines at study entry and over the first 7 days would be independent predictors of death or moderate-severe neurodevelopmental impairment. We also hypothesized that cytokines would be independent predictors of death or moderate-to-severe CP at 6–7 years.

METHODS

This exploratory secondary study was performed in the 15 centers of the Eunice Kennedy Shriver NICHD Neonatal Research Network (NRN) that participated in the Whole Body Hypothermia for Neonates with Hypoxic-ischemic Encephalopathy Trial (11). The cytokines study was approved and commenced enrollment a year following the launch of the main trial; participants were recruited 7/21/2001 to 5/14/2003. Long-term neurodevelopmental follow-up was performed at 6–7 years of age and was completed in August 2010. Infants were eligible for the study if they had severe acidosis or required resuscitation at birth following an acute perinatal event and developed moderate or severe encephalopathy before 6 hours of age (11). Infants were randomly assigned to undergo whole-body hypothermia at 33.5° Celsius for 72 hours or usual care alone. The primary and secondary study were approved by the institutional review boards of each of the participating centers, and written informed consent was obtained from the parent(s) or legal guardian(s).

Cytokine and chemokine measurements

Whole blood spots were collected on filter paper (approximately 0.2mL/day) on days 0–1, 2–4 and 6–7 of life and were dried and subsequently frozen. The stored blood spots were analyzed in duplicate in batches for 24 cytokines and chemokines (including a subset of biomarkers selected because of their predictive ability for brain injury among neonates with encephalopathy and infants who developed cerebral palsy: IL-1β, IL-2, IL-6, IL-8, TNF-α, MCP-1, MIP-1α and RANTES (3–6, 10)). The samples were stored at each participating site at −20°C. Every 3 months, samples from those infants who had completed the study sampling were sent to the Data Center at RTI International. Samples were shipped frozen on dry ice. All samples at RTI were labeled and stored initially at −20°C. The samples were shipped from RTI to an NIH biorepository in 2003–2004 where they were stored at −80°C. Since the original laboratory in Washington DC was unable to run the analyses, samples were shipped to the Statens Serum Institut in Copenhagen, Denmark in 2009. The samples remained frozen at −80°C until analyzed starting in January 2011 after preparing standard curves and controls, with completion of the analysis in April 2011. A multiplex assay using Luminex technology (Luminex Corporation, Austin, Texas) was employed as previously described(12). The majority of the 24 cytokines targeted for these analyses focused on cytokines known to be stable when frozen for up to 23 years(12). To further assess the stability of the analyte concentrations in the present work, additional analyses were completed to assess z-scores of median concentrations by sampling year. Personnel performing laboratory assays were unaware of the neonatal and childhood clinical data.

Neonatal and follow-up data

Clinical data were collected prospectively by trained research coordinators regarding the participant baseline characteristics, maternal and neonatal demographic data, perinatal history and hospital course. All surviving infants were invited to participate in a detailed neurodevelopmental assessment performed at 6–7 years of age that included a neurological examination, ascertainment of neurosensory function, intelligence quotient (IQ) testing (using the Wechsler Preschool and Primary Scale of Intelligence-III (WPPSI-III) or Wechsler Intelligence Scale for Children-IV (WISC-IV)) and assessment of executive function (using the Developmental Neuropsychological Assessment, NEPSY). Details of the study definitions and neurodevelopmental assessments have been published previously (13). Briefly, neurodevelopmental assessments were performed by certified examiners who were masked to cytokine data and treatment group assignment (hypothermia versus usual care). Moderate-severe impairment was defined by any of the following: an IQ score more than two standard deviations below the mean (i.e., <70), cerebral palsy with a Gross Motor Function Classification System (GMFCS) level of III-V (14, 15), bilateral blindness, deafness or refractory epilepsy. No impairment was defined as an IQ score >84 with no CP, hearing or visual impairments, or on-going seizures beyond the neonatal period.

STATISTICAL ANALYSES

Participants with and without cytokine values were compared for baseline and maternal characteristics. The primary analyses were performed for the combined outcome of death or moderate-severe impairment at 6–7 years of age using clinical data and blood spot protein levels collected on days 0–1, 2–4 and 6–7. In addition, pre-specified secondary analyses were performed for the combined outcome of death or cerebral palsy. Due to the skewness of the data, nonparametric Wilcoxon rank sum tests were used to analyze the unadjusted association of cytokine/chemokine levels and death or moderate-severe impairment and death or CP. The Wilcoxon rank sum test was used to compare neonates with moderate-severe NE treated with hypothermia to those who were in the usual care group. To describe associations between post-natal day-specific protein biomarker concentrations and outcomes, we used multinomial logistic regression to estimate odds ratios with 95% confidence intervals for the targeted subset of pre-specified cytokines and chemokines previously associated with brain injury (IL-1β, IL-2, IL-6, IL-8, TNF-α, RANTES, MCP-1 and MIP-1α); models were adjusted for center (as a random effect), treatment group, sex, race and level of HIE at <6 hours of age. Adjustment for multiple comparisons was not performed(16). However, at α=0.05, we would expect 1 out of every 20 comparisons to be significant by chance assuming independent associations with outcome. Many of the cytokines analyzed had values at the upper and lower limits of detection. In these cases, the value of the limit was assigned to minimize the occurrence of missing data. Changes in cytokine concentrations in infants in the hypothermia and control groups and in infants with and without death or moderate-severe impairment were plotted and analyzed over time.

RESULTS

This secondary study commenced enrollment a year following the main trial; parents of 109 participants, among 208 main trial participants, provided informed consent. Cytokine and chemokine blood spots were collected from 56 neonates randomized to hypothermia and 53 neonates randomized to intensive care alone (Figure 1). Baseline maternal and neonatal characteristics are shown in Table 1 and were similar between neonates enrolled in the cytokines secondary study and neonates not enrolled in the cytokines secondary study. 81 (74%) of infants in the cytokines study were born by emergency Cesarean-section, and 102 (94%) required delivery room intubation and continued resuscitation at 10-minutes of life. Mean cord pH was 6.9 ± 0.2 and mean base deficit was 17.9 ± 7.8 mmol/liter. On neurological examination, 65% of neonates had moderate encephalopathy and 35% had severe encephalopathy at enrollment. 6–7 year follow-up data were available for 99 of 109 (91%) participants. Serial blood protein data (2 or more samples) were available for 103 (94%) surviving neonates. At 6–7 years of age, 54 (55%) infants developed the primary outcome of death or moderate-severe neurodevelopmental impairment, 30 (30%) died, and 42 (42%) died or developed moderate-severe CP.

Figure 1.

Figure 1.

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Flow diagram of participants.

Table 1.

Maternal and neonatal baseline characteristics for neonates with and without cytokine blood samples

Characteristic* Cytokine blood samples (N=109) No samples (N=99) P valueǂ
Maternal
Race1 - N (%) 0.51
 Black 41 (38%) 31 (31%)
 White 38 (35%) 34 (34%)
 Other 30 (28%) 34 (34%)
Maternal age years 27.3 ± 5.8 27.3 ± 6.4 0.86
Married - N (%) 60/106 (57%) 55/97 (57%) 1.00
Education less than high school - N (%) 29/83 (35%) 25/71 (35%) 1.00
Gravida - median no. (Q1, Q3) 2 (1, 4) 2 (1, 3) 0.71
   - range 1–9 1–10
Parity - median no. (Q1, Q3) 2 (1, 3) 2 (1, 3) 0.66
   - range 1–8 1–7
Complications of pregnancy - N (%)
 Chronic hypertension 14 (13%) 12 (12%) 1.00
 Antepartum hemorrhage 16 (15%) 14 (14%) 1.00
 Thyroid disease 1 (1%) 1 (1%) 1.00
 Diabetes 9 (8%) 8 (8%) 1.00
 Chorioamnionitis N/A N/A N/A
Intrapartum complications - N (%)
 Fetal HR decelerations 80/109 (73%) 73/98 (74%) 0.88
 Cord prolapse 18 (17%) 19 (19%) 0.72
 Uterine rupture 13 (12%) 16 (16%) 0.43
 Maternal pyrexia 9/109 (8%) 12/98 (12%) 0.37
 Shoulder dystocia 10/108 (9%) 10/99 (10%) 1.00
 Maternal hemorrhage 8 (7%) 6 (6%) 0.79
Any intrapartum complications - N (%) 95 (87%) 87 (88%) 1.00
Any intrapartum complications, not including fetal HR decelerations - N (%) 51/108 (47%) 54/98 (55%) 0.27
Labor - hours 11.7 ± 7.3 12.8 ± 11.2 0.90
Labor - hours (including no labor as 0) 8.1 ± 8.1 7.5 ± 10.6 0.18
Rupture of membranes - hours 6.6 ± 8.9 5.2 ± 11.7 0.13
Emergency cesarean delivery - N (%) 81 (74%) 71 (72%) 0.75
Neonatal
Gestational age - median no. (Q1, Q3) 39 (38, 40) 39 (38, 40) 0.78
   - range 36–42 36–43
Age at randomization - hours 4.3 ± 1.3 4.3 ± 1.2 0.76
Transferred from birth hospital - N (%) 48 (44%) 45 (45%) 0.89
Male sex - N (%) 63 (58%) 54 (55%) 0.68
Apgar score ≤ 5 - N (%)
 At 5 minute 97/108 (90%) 92/99 (93%) 0.47
 At 10 minute 77/101 (76%) 77/90 (86%) 0.14
Birth weight - grams 3369 ± 690 3386 ± 560 0.34
   - range 2100–5970 2035–4994
Length - cm 50.8 ± 3.0 50.8 ± 3.3 0.76
   - range 43–57 40–58
Head circumference – cm 34.3 ± 1.7 34.1 ± 1.5 0.36
   - range 30–38.8 30.5–37
Intubation in delivery room - N (%) 102 (94%) 93 (94%) 1.00
Continued resuscitation at 10 minutes - N (%) 102 (94%) 93 (94%) 1.00
Time to spontaneous respiration ≥ 10 minutes – N (%) 69/102 (68%) 71/95 (75%) 0.35
Cord blood
 pH 6.9 ± 0.2 6.8 ± 0.2 0.09
 Base deficit –mmol/liter 17.9 ± 7.8 20.7 ± 7.5 0.07
Seizures at baseline – N (%) 46 (42%) 48 (48%) 0.40
Moderate encephalopathy – N (%)2 71/109 (65%) 63/98 (64%) 1.00
Severe encephalopathy – N (%)2 38/109 (35%) 35/98 (36%)
Inotropic support at baseline – N (%) 37/109 (34%) 22/98 (22%) 0.09
Anticonvulsants at baseline – N (%) 43/99 (43%) 40/90 (44%) 1.00
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*

Plus-minus values are means ±SD. Percentages are based on the numbers of mothers or neonates for whom data were available.

ǂ

Fisher’s exact test

1

Race was determined by interviewing the mothers.

2

Data are for this characteristic at the time of enrollment.

N, number; HR, heart rate; cm, centimeters.

Stability of protein levels was assessed by calculating z-scores for all analytes by year of specimen collection; no significant difference in median protein concentrations was detected for any of the blood proteins tested (Figure 2 online).

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Z-scores of median analyte concentrations by birth year

Univariate analyses of blood protein levels and the risk of poor outcomes

Infants who developed the primary outcome (death/impairment) had significantly lower median Regulated upon Activation Normal T-Cell Expressed and Secreted (RANTES) or CCL5 on day 0–1 (p=0.02) and lower neurotrophic factor 4 (NT-4) levels (p=0.04) as compared with neonates without adverse outcome (Table 2). Day 3 cytokine and chemokine levels revealed a similar pattern for NT-4 levels between the death and impairment group and the no death or impairment group. On day 7, C-reactive protein (p= 0.04) and monocyte chemotactic protein-1 (MCP-1) (p=0.02) were elevated among neonates with death/impairment as compared to neonates who survived without impairment. Higher day 0–1 levels of BDNF, CRP and TNF-α were observed among neonates who died or developed cerebral palsy (Table 2). Full data for all 24 analytes is available on-line for outcomes of death/neurodevelopmental impairment and death/CP (Tables 3 and 4).

Table 2.

Comparison of median cytokines on days 0–7 among infants with adverse 6–7 year outcome and no adverse outcome depicting only significant biomarkers (among 24 analytes)

Outcome
Analyte measured Death or Moderate-severe impairment (N=54) No death or impairment (N=45) P value Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
Day 0–1 N=54 N=43
NT-4 (pg/ml)* 11.8 (7.5, 23.5) 18.0 (11.8, 26.0) 0.04
RANTES (ng/ml) 73.7 (43.0, 120.0) 111.0 (70.1, 138.9) 0.02
Day 2–4 N=43 N=45
IL-5 (pg/ml) 4.0 (4.0, 4.0) 4.0 (4.0, 11.8) 0.02
NT-4 (pg/ml)* 13.8 (8.6, 28.4) 20.5 (11.5, 45.6) 0.048
Day 6–7 N=29 N=33
CRP (mg/L)* 1.3 (0.5, 2.6) 0.6 (0.3, 1.1) 0.04
MCP 1 (pg/ml) 1520.6 (727.5, 2679.0) 803.7 (421.6, 1316.2) 0.02
Death or moderate-severe CP (N=42) No death or moderate-severe CP (N=57)
Median (Q1, Q3) Median (Q1, Q3)
Day 0–1 N=42 N=55
BDNF (ng/ml)* 8.4 (4.6, 13.7) 5.5 (2.8, 8.8) 0.02
CRP (mg/L)* 0.9 (0.4, 1.6) 0.5 (0.2, 1.3) 0.04
TNF-alpha 29.8 (13.4, 50.5) 15.1 (4.0, 41.0) 0.03
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Q, quartile; NT-4, Neurotrophic factor-4; RANTES, Regulated upon Activation Normal T-cell Expressed and Secreted; IL, interleukin; CRP, C-reactive protein; MCP1, monocyte chemotactic protein 1; BDNF, Brain-derived neurotrophic factor; TNF-alpha, tumor necrosis factor-alpha

*

DBSS concentrations may decrease over time when stored for prolonged periods.

Full data for all 24 analytes is available on-line for outcomes of death/neurodevelopmental impairment and death/CP (tables 3 and 4)

Table 3.

Comparison of median cytokines on days 0–7 among infants with death or impairment and no death or impairment at 6–7 years.

Table 3A – Initial cytokines (median values of samples from days 0–1)
Analyte measured Death or Mod-severe impairment (N=54)
N=54 day 0–1		 No death or impairment (N=45)
N=43 day 0–1		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL-1beta* 64.1 (16.5, 98.0) 68.9 (31.3, 112.0) 0.45
IL-2 4.0 (4.0, 9.0) 4.0 (4.0, 6.2) 0.14
IL-4 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.92
IL-5 4.0 (4.0, 4.0) 4.0 (4.0, 6.6) 0.42
IL-6 126.2 (25.8, 642.0) 101.8 (19.7, 1219.0) 0.56
IL-8* 3986.5 (1748.5, 4000.0) 2588.2 (1573.5, 4000.0) 0.13
IL-10 646.5 (289.7, 920.0) 409.9 (257.0, 631.5) 0.08
IL-12 4.0 (4.0, 33.0) 10.4 (4.0, 25.1) 0.69
IL-17 15.0 (4.0, 33.1) 14.4 (4.0, 28.0) 0.62
IL-18 (ng/ml) 4.1 (2.8, 6.3) 4.3 (3.1, 7.3) 0.61
IL-6ra (ng/ml) 55.2 (39.7, 91.2) 70.2 (52.8, 104.3) 0.06
BDNF (ng/ml)* 6.2 (2.9, 12.0) 6.5 (3.2, 9.6) 0.86
CRP (mg/L)* 0.8 (0.3, 1.6) 0.7 (0.2, 1.3) 0.11
GM-CSF 34.5 (15.9, 67.1) 41.8 (10.0, 70.6) 0.86
IFN-gamma 4.0 (4.0, 9.5) 4.0 (4.0, 9.8) 0.61
NT-4* 11.8 (7.5, 23.5) 18.0 (11.8, 26.0) 0.04
TGF-beta 1401.6 (827.0, 1839.0) 1294.4 (869.2, 1911.4) 0.88
TNF-alpha 25.7 (4.0, 49.5) 19.5 (4.0, 41.0) 0.24
TNF-beta 29.4 (10.0, 126.9) 35.3 (10.0, 118.4) 0.93
TREM-1* 488.0 (488.0, 1235.7) 488.0 (488.0, 1438.0) 0.79
RANTES (ng/ml) 73.7 (43.0, 120.0) 111.0 (70.1, 138.9) 0.02
MIP-1 alpha 1075.4 (523.4, 2089.7) 1133.1 (749.6, 2138.0) 0.40
MCP 1 2243.7 (718.5, 5946.5) 1433.5 (891.7, 3907.9) 0.80
MMP-9 (ug/ml)* 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 0.26
All units are pg/ml, except as indicated.
Table 3B – Day 3 cytokines (median values of samples days 2–4)
Analyte measured Death or Mod-severe impairment (N=54)
N=43 on day 2–4		 No death or impairment (N=45)
N=45 on day 2–4		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL-1beta* 43.8 (4.0, 77.7) 45.0 (4.0, 90.1) 0.79
IL-2 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.96
IL-4 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.66
IL-5 4.0 (4.0, 4.0) 4.0 (4.0, 11.8) 0.02
IL-6 30.4 (4.0, 107.0) 22.4 (4.0, 103.0) 0.90
IL-8* 1034.0 (471.0, 2943.5) 700.7 (278.9, 1811.0) 0.09
IL-10 201.1 (132.7, 335.0) 178.6 (115.0, 289.6) 0.49
IL-12 8.5 (4.0, 29.1) 18.5 (4.0, 36.2) 0.19
IL-17 15.0 (4.0, 36.7) 11.8 (4.0, 31.4) 0.78
IL-18 (ng/ml) 3.3 (2.5, 4.6) 4.1 (2.6, 6.0) 0.12
IL-6ra (ng/ml) 52.6 (39.1, 81.0) 63.9 (53.8, 83.1) 0.13
BDNF (ng/ml)* 4.5 (1.9, 7.2) 4.8 (2.3, 7.2) 0.57
CRP (mg/L)* 1.5 (0.8, 2.6) 1.2 (0.7, 1.9) 0.19
GM-CSF 28.6 (10.0, 66.0) 33.4 (10.0, 85.6) 0.28
IFN-gamma 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.71
NT-4* 13.8 (8.6, 28.4) 20.5 (11.5, 45.6) 0.048
TGF-beta 892.8 (404.6, 1382.9) 1016.0 (575.0, 1461.5) 0.39
TNF-alpha 26.1 (4.0, 48.9) 11.9 (4.0, 33.8) 0.06
TNF-beta 71.3 (10.0, 130.8) 70.2 (10.0, 166.0) 0.97
TREM-1* 488.0 (488.0, 1234.5) 488.0 (488.0, 1177.3) 0.44
RANTES (ng/ml) 53.5 (35.3, 119.9) 93.6 (57.7, 129.0) 0.07
MIP-1 alpha 797.9 (461.2, 1431.0) 998.0 (544.3, 1754.8) 0.62
MCP 1 1195.5 (588.4, 5834.0) 1552.2 (838.3, 2946.5) 0.97
MMP-9 (ug/ml)* 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 0.97
All units are pg/ml, except as indicated. Some infants had multiple samples done within the window (2–4 days); these results were averaged.
Table 3C – Day 7 cytokines (median values of samples days 6–7)
Analyte measured Death or Mod-severe impairment (N=54)
N=29 on day 6–7		 No death or impairment (N=45)
N=33 on day 6–7		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL-1beta* 32.0 (4.0, 106.0) 59.7 (13.0, 103.9) 0.72
IL-2 4.0 (4.0, 11.7) 4.0 (4.0, 4.0) 0.07
IL-4 4.0 (4.0, 9.0) 4.0 (4.0, 4.0) 0.56
IL-5 11.7 (4.0, 20.2) 9.0 (4.0, 13.3) 0.40
IL-6 15.1 (4.0, 70.0) 4.0 (4.0, 34.8) 0.30
IL-8* 445.1 (109.9, 839.0) 374.0 (158.0, 528.7) 0.73
IL-10 232.1 (125.0, 310.7) 146.8 (101.6, 247.0) 0.25
IL-12 11.4 (4.0, 42.9) 20.0 (4.0, 36.1) 0.63
IL-17 18.3 (4.0, 29.6) 22.9 (4.0, 33.1) 0.92
IL-18 (ng/ml) 3.4 (2.2, 5.4) 3.1 (2.2, 5.4) 0.88
IL-6ra (ng/ml) 53.0 (41.6, 86.9) 69.8 (56.7, 87.2) 0.18
BDNF (ng/ml)* 7.2 (3.2, 8.9) 7.0 (4.4, 12.8) 0.29
CRP (mg/L)* 1.3 (0.5, 2.6) 0.6 (0.3, 1.1) 0.04
GM-CSF 29.5 (10.0, 71.6) 33.0 (10.0, 66.5) 0.95
IFN-gamma 4.0 (4.0, 12.3) 4.0 (4.0, 11.5) 1.00
NT-4* 14.2 (7.5, 28.0) 19.0 (11.0, 28.9) 0.65
TGF-beta 1422.7 (587.6, 1908.0) 1546.1 (1051.0, 2035.0) 0.54
TNF-alpha 26.0 (4.0, 56.8) 16.7 (4.0, 38.9) 0.25
TNF-beta 89.5 (41.0, 176.0) 81.0 (10.0, 201.3) 0.40
TREM-1* 488.0 (488.0, 1020.2) 488.0 (488.0, 488.0) 0.54
RANTES (ng/ml) 71.8 (41.2, 129.6) 116.5 (73.6, 160.0) 0.11
MIP-1 alpha 755.0 (634.1, 1213.5) 899.2 (554.0, 1413.0) 0.94
MCP 1 1520.6 (727.5, 2679.0) 803.7 (421.6, 1316.2) 0.02
MMP-9 (ug/ml)* 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 1.00
All units are pg/ml, except as indicated.
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Q, quartile; IL, interleukin; BDNF, Brain-derived neurotrophic factor; CRP, C-reactive protein; GM-CSF, Granulocyte-macrophage colony-stimulating factor; IFN-gamma, Interferon-gamma; NT4, Neurotrophic factor-4; TGF-beta, Transforming growth factor-beta; TNF-alpha, tumor necrosis factor-alpha; TREM-1, Triggering Receptor Expressed On Myeloid Cells 1; RANTES, Regulated upon Activation Normal T-cell Expressed and Secreted; MIP-1 alpha, macrophage inflammatory protein 1 alpha; MCP1, monocyte chemotactic protein-1; MMP-9, Matrix metallopeptidase 9

*

Concentrations may decrease over time when stored for prolonged periods.

Table 4.

Comparison of median cytokines on days 0–7 among infants with death or moderate-severe CP and no death or moderate-severe CP.

Table 4A – Initial cytokines (median values of samples from day 0–1)
Analyte measured Death or moderate-severe CP (N=42)
N=42 on day 0–1		 No death or moderate-severe CP (N=57)
N=55 on day 0–1		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL1 beta* 66.8 (34.0, 98.0) 47.0 (19.3, 109.6) 0.68
IL-2 4.0 (4.0, 9.0) 4.0 (4.0, 6.2) 0.10
IL-4 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.65
IL-5 4.0 (4.0, 5.9) 4.0 (4.0, 6.0) 0.81
IL-6 104.9 (24.0, 1235.0) 117.3 (25.8, 703.0) 0.95
IL-8* 3792.6 (1748.5, 4000.0) 3163.4 (1584.0, 4000.0) 0.57
IL-10 655.4 (246.7, 1014.6) 444.0 (270.3, 688.0) 0.28
IL-12 8.7 (4.0, 33.0) 6.6 (4.0, 20.1) 0.44
IL-17 18.9 (4.0, 36.3) 9.6 (4.0, 27.3) 0.13
IL-18 (ng/ml) 4.2 (2.8, 6.5) 4.3 (3.0, 6.8) 0.88
IL-6ra (ng/ml) 58.4 (43.6, 95.5) 64.6 (48.6, 104.3) 0.38
BDNF (ng/ml)* 8.4 (4.6, 13.7) 5.5 (2.8, 8.8) 0.02
CRP (mg/L)* 0.9 (0.4, 1.6) 0.5 (0.2, 1.3) 0.04
GM-CSF 31.8 (10.0, 71.2) 41.8 (10.0, 69.4) 0.87
IFN-gamma 4.0 (4.0, 9.0) 4.0 (4.0, 10.3) 0.84
NT-4* 14.1 (8.5, 23.8) 14.6 (8.8, 24.8) 0.88
TGF-beta 1483.8 (936.7, 1951.4) 1229.8 (781.2, 1840.0) 0.19
TNF-alpha 29.8 (13.4, 50.5) 15.1 (4.0, 41.0) 0.03
TNF-beta 44.2 (10.0, 167.9) 24.9 (10.0, 97.4) 0.22
TREM-1* 758.2 (488.0, 1329.8) 488.0 (488.0, 1365.9) 0.65
RANTES (ng/ml) 80.8 (47.9, 126.7) 104.6 (64.6, 129.4) 0.29
MIP-1 alpha 1227.4 (519.3, 2249.7) 1059.8 (727.5, 1993.6) 0.77
MCP 1 2179.8 (656.8, 6177.5) 1638.2 (902.4, 4115.0) 0.92
MMP-9 (ug/ml)* 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 0.92
All units are pg/ml, except as indicated.
Table 4B – Day 3 cytokines (median values of samples from days 2–4)
Analyte measured Death or moderate-severe CP (N=42)
N=31 on day 2–4		 No death or moderate-severe CP (N=57)
N=57 on day 2–4		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL1-beta* 47.3 (22.6, 94.1) 35.6 (4.0, 80.2) 0.35
IL-2 4.0 (4.0, 9.3) 4.0 (4.0, 4.0) 0.40
IL-4 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.39
IL-5 4.0 (4.0, 4.0) 4.0 (4.0, 11.1) 0.10
IL-6 28.0 (4.0, 98.0) 33.1 (4.0, 137.0) 0.58
IL-8* 1034.0 (513.0,2930.2) 741.5 (323.0, 2491.0) 0.21
IL-10 199.6 (112.8, 335.0) 182.8 (132.0, 322.3) 0.93
IL-12 11.9 (4.0, 28.5) 16.6 (4.0, 36.6) 0.43
IL-17 15.3 (4.0, 36.7) 11.8 (4.0, 32.0) 0.67
IL-18 (ng/ml) 3.2 (2.4, 3.8) 4.1 (2.8, 6.9) 0.06
IL-6ra (ng/ml) 59.5 (39.9, 91.7) 61.3 (46.6, 79.2) 0.81
BDNF (ng/ml)* 5.8 (2.8, 7.8) 3.6 (1.8, 7.0) 0.11
CRP (mg/L)* 1.5 (0.8, 2.6) 1.3 (0.7, 2.6) 0.53
GM-CSF 28.6 (10.0, 69.8) 33.4 (10.0, 77.6) 0.44
IFN-gamma 4.0 (4.0, 4.0) 4.0 (4.0, 4.0) 0.95
NT-4* 15.0 (9.9, 25.4) 19.3 (10.6, 43.0) 0.30
TGF-beta 1062.9 (609.0, 1616.7) 915.0 (404.6, 1348.2) 0.12
TNF-alpha 28.2 (4.0, 50.9) 14.0 (4.0, 33.8) 0.14
TNF-beta 89.5 (10.0, 153.0) 48.1 (10.0, 135.9) 0.35
TREM-1* 488.0 (488.0, 1234.5) 488.0 (488.0, 1177.3) 0.49
RANTES (ng/ml) 82.7 (45.4, 127.5) 73.6 (41.8, 120.9) 0.75
MIP-1 alpha 843.0 (453.0, 1450.0) 961.6 (549.5, 1701.4) 0.65
MCP 1 1071.0 (447.0, 3099.0) 1660.7 (955.2, 3364.0) 0.16
MMP-9 (ug/ml)* 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 0.66
All units are pg/ml, except as indicated. Some infants had multiple samples done within the window (2–4 days); these results were averaged.
Table 4C – Day 7 cytokines (median values of samples from days 6–7)
Analyte measured Death or moderate-severe CP (N=42)
N=21 on day 6–7		 No death or moderate-severe CP (N=57)
N=41 on day 6–7		 P value
Wilcoxon-Mann-Whitney
Median (Q1, Q3) Median (Q1, Q3)
IL1-beta* 54.8 (4.0, 106.0) 59.7 (13.0, 103.9) 0.74
IL-2 4.0 (4.0, 12.2) 4.0 (4.0, 4.0) 0.06
IL-4 4.0 (4.0, 9.9) 4.0 (4.0, 4.0) 0.21
IL-5 11.7 (4.0, 18.7) 11.0 (4.0, 13.4) 0.73
IL-6 15.1 (4.0, 57.0) 8.3 (4.0, 48.3) 0.58
IL-8* 227.2 (109.9, 648.0) 383.9 (158.0, 728.9) 0.60
IL-10 232.5 (147.2, 373.0) 153.0 (103.3, 249.0) 0.10
IL-12 29.4 (4.0, 48.3) 19.0 (4.0, 31.4) 0.14
IL-17 25.0 (4.0, 37.0) 17.7 (4.0, 30.0) 0.32
IL-18 (ng/ml) 2.8 (2.2, 6.1) 3.5 (2.4, 5.4) 0.85
IL-6ra (ng/ml) 74.4 (45.4, 101.2) 64.6 (46.7, 81.1) 0.69
BDNF (ng/ml)* 8.3 (5.9, 10.7) 5.9 (4.0, 10.4) 0.23
CRP (mg/L*) 1.8 (0.4, 2.6) 0.7 (0.3, 1.3) 0.24
GM-CSF 30.6 (10.0, 97.0) 24.0 (10.0, 58.9) 0.26
IFN-gamma 8.6 (4.0, 14.1) 4.0 (4.0, 11.5) 0.36
NT-4* 22.1 (8.5, 33.1) 15.3 (8.0, 27.1) 0.42
TGF-beta 1695.6 (1319.2, 2310.6) 1269.4 (678.2, 1767.3) 0.09
TNF-alpha 26.0 (15.6, 58.5) 16.7 (4.0, 44.0) 0.17
TNF-beta 115.5 (66.7, 202.4) 81.0 (10.0, 201.3) 0.12
TREM-1* 488.0 (488.0, 1087.9) 488.0 (488.0, 1020.2) 0.47
RANTES (ng/ml) 89.7 (51.2, 156.6) 100.0 (60.7, 157.9) 0.79
MIP-1 alpha 842.0 (648.9, 1286.0) 793.7 (554.0, 1199.0) 0.38
MCP 1 1235.6 (688.0, 1892.5) 934.0 (537.0, 1607.8) 0.41
MMP-9*(ug/ml) 0.5 (0.5, 0.5) 0.5 (0.5, 0.5) 1.0
All units are pg/ml, except as indicated.
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Q, quartile; IL, interleukin; BDNF, Brain-derived neurotrophic factor; CRP, C-reactive protein; GM-CSF, Granulocyte-macrophage colony-stimulating factor; IFN-gamma, Interferon-gamma; NT4, Neurotrophic factor-4; TGF-beta, Transforming growth factor-beta; TNF-alpha, tumor necrosis factor-alpha; TREM-1, Triggering Receptor Expressed On Myeloid Cells 1; RANTES, Regulated upon Activation Normal T-cell Expressed and Secreted; MIP-1 alpha, macrophage inflammatory protein 1 alpha; MCP1, monocyte chemotactic protein-1; MMP-9, Matrix metallopeptidase 9

*

Concentrations may decrease over time when stored for prolonged periods.

Multivariable regression analysis of log-transformed cytokine values and outcome

On multivariable regression analysis adjusted for center (as a random effect), treatment group, sex, race, and level of initial HIE, two chemokines and one cytokine of the pre-specified protein biomarkers were associated with 6–7 year outcomes (Table 5). Higher early RANTES levels were inversely associated with death/impairment (OR 0.31, 95% CI: 0.13–0.74), while day 7 MCP-1 levels were directly associated with death/impairment (OR 3.70, 95% CI: 1.42–9.61). Day 0–1 TNFα levels were associated with increased odds of death or moderate-severe cerebral palsy (OR 1.66, 95% CI: 1.10–2.51). Cytokine data improved prediction of poor outcomes as compared with a multivariable model that included center, treatment group, sex, race and HIE level alone (as demonstrated by the area under the ROC curves, also shown in Table 5). Data with all 24 possible cytokines from the in-house multiplex assay included in the multivariable regression model revealed similar results. CRP on day 7 also was associated with poor outcomes (OR 2.08, 95% CI: 1.09–3.94).

Table 5.

Multivariable regression analysis for components of death or moderate/severe impairment, death or moderate/severe CP

For death or moderate-severe impairment
Initial (days 0–1) Day 2–4 Day 6–7
Cytokine/Chemokine OR (95% CI) p-value AUC OR (95% CI) p-value AUC OR (95% CI) p-value AUC
IL-1beta* 0.85 (0.59–1.21) 0.36 0.68 0.94 (0.66–1.35) 0.73 0.71 0.96 (0.64–1.42) 0.82 0.77
IL-2 2.02 (0.92–4.42) 0.08 0.73 0.94 (0.43–2.09) 0.89 0.71 2.00 (0.79–5.12) 0.14 0.82
IL-6 1.07 (0.88–1.30) 0.50 0.69 0.95 (0.72–1.24) 0.69 0.71 1.20 (0.82–1.75) 0.34 0.79
IL-8* 1.14 (0.68–1.90) 0.62 0.68 1.27 (0.86–1.87) 0.23 0.74 1.21 (0.84–1.75) 0.31 0.80
TNF-alpha 1.31 (0.90–1.92) 0.16 0.69 1.43 (0.94–2.16) 0.09 0.73 1.42 (0.85–2.36) 0.18 0.82
RANTES 0.31 (0.13–0.74) 0.009 0.75 0.47 (0.24–0.92) 0.03 0.77 0.50 (0.19–1.36) 0.17 0.79
MCP-1 1.17 (0.76–1.79) 0.47 0.70 1.40 (0.83–2.35) 0.20 0.75 3.70 (1.42–9.61) 0.008 0.86
NT-4 * 0.56 (0.31–1.01) 0.056 0.72 0.56 (0.32–0.99) 0.05 0.76 0.82 (0.42–1.60) 0.55 0.76
MIP-1alpha 0.81 (0.53–1.24) 0.32 0.69 0.92 (0.59–1.44) 0.71 0.71 0.98 (0.56–1.71) 0.93 0.78
No cytokines N/A N/A 0.67 N/A N/A 0.71 N/A N/A 0.78
For death or moderate-severe CP
Initial (days 0–1) Day 2–4 Day 6–7
Cytokine/Chemokine OR (95% CI) p-value AUC OR (95% CI) p-value AUC OR (95% CI) p-value AUC
IL-1beta* 1.04 (0.72–1.50) 0.83 0.72 1.17 (0.78–1.76) 0.44 0.75 0.89 (0.57–1.38) 0.59 0.83
IL-2 1.81 (0.87–3.78) 0.11 0.74 1.06 (0.47–2.40) 0.88 0.74 1.82 (0.72–4.60) 0.20 0.85
IL-6 1.00 (0.81–1.22) 0.97 0.72 0.90 (0.67–1.20) 0.46 0.74 1.14 (0.77–1.69) 0.52 0.82
IL-8* 0.90 (0.52–1.54) 0.68 0.72 1.33 (0.85–2.11) 0.21 0.76 0.94 (0.65–1.37) 0.76 0.82
TNF-alpha 1.66 (1.10–2.51) 0.02 0.77 1.36 (0.88–2.13) 0.17 0.77 1.41 (0.82–2.41) 0.21 0.86
RANTES 0.51 (0.23–1.12) 0.09 0.74 1.00 (0.47–2.11) 1.00 0.73 1.01 (0.36–2.87) 0.98 0.82
MCP-1 1.03 (0.67–1.58) 0.91 0.72 0.91 (0.54–1.52) 0.70 0.74 1.88 (0.81–4.38) 0.14 0.84
NT-4* 0.98 (0.55–1.74) 0.94 0.72 0.81 (0.47–1.41) 0.45 0.74 1.39 (0.66–2.90) 0.38 0.84
MIP-1alpha 0.87 (0.57–1.32) 0.51 0.72 1.01 (0.59–1.70) 0.98 0.74 1.23 (0.71–2.15) 0.45 0.85
No cytokines N/A N/A 0.72 N/A N/A 0.74 N/A N/A 0.81
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Cytokines are log-transformed; models also adjust for center (as a random effect), treatment group, sex, race and level of HIE at <6 hours of age.

OR, odds ratio; AUC, area under the curve; IL, interleukin; TNF-alpha, tumor necrosis factor-alpha; RANTES, Regulated upon Activation Normal T-cell Expressed and Secreted; MCP-1, monocyte chemotactic protein-1; MMP-9, Matrix metallopeptidase 9; NT-4, Neurotrophic factor-4; MIP-1 alpha, macrophage inflammatory protein 1 alpha.

*

Concentrations may decrease over time when stored for prolonged periods.

Longitudinal profile of cytokines among neonates with and without subsequent death or impairment

Figure 3 reveals the median chemokine/cytokine values of RANTES and MCP1 at each time point assessed among neonates with and without death or moderate-severe impairment (online-only). Levels of pro-inflammatory cytokines IL-1β, IL-2, IL-6, IL-8, tumor necrosis factor-α and MIP-1α showed no association with the composite outcome of death or impairment at any time point. No statistically significant difference in median cytokine levels was observed by treatment group (hypothermia vs. usual care) for IL-1β, IL-2, IL-6, IL-8, TNF-α, RANTES, and MIP-1α at each time point studied; MCP-1 levels remained higher for a longer duration among neonates exposed to hypothermia (Figure 4 online-only).

Figure 3.

Figure 3.

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Boxplots depicting median RANTES and MCP1 levels on days 0–7 among neonates with (black) and without (gray) death or moderate-severe impairment. *Asterisks denote statistically significant differences.

RANTES, Regulated upon Activation Normal T-cell Expressed and Secreted; MCP-1, monocyte chemotactic protein-1; N, no; Y, yes.

Figure 4.

Figure 4.

Figure 4.

Figure 4.

Figure 4.

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Median cytokine values (solid lines) and interquartile bounds (dotted lines, Q1 and Q3) for IL-1β, IL-2, IL-6, IL-8, TNF-α, MCP-1, MIP-1α and RANTES among patients treated with hypothermia versus normothermia at 0–1, 2–4 and 6–7 days. Hypothermia participants are depicted in blue and normothermia participants are depicted in red. The p-values shown are from Wilcoxon tests.

DISCUSSION

Preclinical and clinical data suggest that cytokines, chemokines and damage-associated proteins are released following acute hypoxic-ischemic brain injury as may be seen in some cases of neonatal encephalopathy (6, 7, 10, 17–33). These protein biomarkers have neuropoietic effects that may influence brain injury, pathogenesis and subsequent neuronal development and synaptic signaling (34, 35). In this secondary study, we profiled the cytokine/chemokine response from day 0–7 in a subgroup of 109 participants of the NICHD Neonatal Research Network Trial of Whole Body Hypothermia and examined the relationship between cytokines, chemokines and damage-associated proteins and 6–7 year neurodevelopmental outcomes. 24 analytes were examined at study entry and over the first 7 days of life using DBSS. The principle findings of our study were that higher early RANTES was associated with reduced odds of death/impairment, while day 6–7 MCP-1 was associated with increased odds of death/impairment at 6–7 years suggesting that these immune modulating proteins may play an important role in the cascade of brain injury associated with some cases of neonatal encephalopathy.

RANTES (or CCL5) is a potent leukocyte activator implicated in multiple inflammatory and neurological disorders. The relationship between RANTES levels and brain injury is complex. Studies report both increased and decreased RANTES levels in association with poor outcomes. In a small case-control study of 31 children with spastic diplegia, Nelson reported elevated RANTES levels on dried neonatal blood spots among cases as compared with controls; no difference in levels was reported among children with spastic quadriplegia and controls (10). Select studies on neuroinflammatory and neurodegenerative disorders report an association between elevated RANTES levels and aberrant inflammatory T cell migration (36), synaptic excitability and neuroinflammation (37). RANTES’ association with synaptic excitability, may be important to neuronal signaling and may lead to significant downstream consequences.

Preclinical models suggest a neuroprotective role for RANTES as well. Among cell cultures preincubated with RANTES, there is improved neuronal cell survival (38) and suppression of microglial neurotoxicity (suppression of LPS induced IL-1β, IL-6, TNFα and inducible nitric oxide synthase) (39). Furthermore, pharmacological induction of RANTES in vivo has been shown to prevent gp120 mediated neuronal injury (important in HIV associated cognitive impairment) (40). Recently, Valerio et al. reported that RANTES activates several genes involved in neuronal differentiation, survival and synaptogenesis further supporting a putative neuroprotective role (41). We found elevated RANTES levels among our intact survivors. High RANTES levels early in the disease course may mediate outcomes in neonates with encephalopathy. The causal pathway that leads to neonatal encephalopathy may be multifactorial. Neuroinflammation and synaptic excitability may serve as important therapeutic targets for some neonates with altered chemokine/receptor levels; for others, alternative strategies may be necessary. Pharmacological induction of RANTES with morphine has been shown to reduce neuronal degeneration and neurocognitive impairment in HIV associated neuroinflammation (40). If substantiated, a similar strategy may be considered for HIE.

The second chemokine associated with 6–7year outcomes was MCP-1; MCP-1 (CCL2) is best known for its chemotactic activity for monocytes, basophils and immature dendritic cells. Additionally, it is expressed by neurons, microglia and astrocytes in the presence of neuro-inflammation. MCP-1 concentrations are increased in brain ischemia (42–44), epilepsy (45–48), and neurodegenerative disorders (49, 50). In children, peripheral blood MCP-1 levels have been associated with cerebral palsy (10) and autism spectrum disorders (51). In our study, day 7 MCP-1 levels were directly associated with increased odds of death/impairment. Once again, MCP-1 may be both a biomarker and a therapeutic target. In population based prospective cohort studies, baseline circulating MCP-1 levels in stroke-free individuals were associated with long-term stroke risk. In fact, the results followed a dose-response pattern: higher baseline MCP-1 levels were associated with a higher risk of subsequent ischemic stroke in patients followed for a mean interval of 16.3 years. MCP-1 inhibition might represent a feasible therapeutic target to minimize inflammatory brain injury. Presently, Phase I/II clinical trials are underway for other inflammatory diseases, atherosclerosis and stroke (52).

Since the introduction of therapeutic hypothermia, limited data are available on the association between candidate serum biomarkers and neurodevelopmental outcomes among neonates with presumptive NE (7, 8). These studies (including our own) are limited by small sample size, lack of controls without neonatal encephalopathy, differences in the types and timing of biological specimens, bioassays performed and differences in the stringency of definitions of neurodevelopmental outcomes. Jenkins et al. reported cytokine data from their randomized, controlled phase II trial of hypothermia (33°C for 48h) in which cytokines were measured in 28 hypothermic and 22 normothermic neonates (7). Elevated IL-6 and MCP-1 levels within 9 hours after birth and low MIP1-α levels at 60–70 hours of age were associated with death or severe neurodevelopmental impairment at 12 months. IL-6, IL-8 and MCP-1 levels among neonates in the hypothermia group revealed a biphasic pattern with early and late peaks. Our study supports persistently elevated MCP-1 levels at 6–7 days. Chalak et al assessed the association between protein biomarkers and 15–18 month neurodevelopmental outcomes in a prospective cohort study of 27 neonates diagnosed with HIE who were treated with hypothermia if moderate-severe encephalopathy was present (n=20) (8). Elevated concentrations of IL-1, IL-6, IL-8, glial fibrillary acidic protein and vascular endothelial growth factor obtained from umbilical arterial samples at 6–24 hours were associated with abnormal neurodevelopmental outcomes (defined by a Bayley Scales of Infant and Toddler Development-III composite score <85 on any domain, cerebral palsy, blindness or deafness). Serial cytokine levels were unaffected by therapeutic hypothermia or rewarming, similar to our study. We found no association between IL-1, IL-6 and IL-8 levels and 6–7 year neurodevelopmental outcomes; however, timing of specimen collection and definition of neurodevelopmental outcomes differed. IL-1 and IL-8 in particular are known to exhibit some biological degradation in frozen DBSS over time. Hypothermia did not alter RANTES and MCP1 in our study; the depth of cooling and the timing of our samples may have contributed to the lack of alteration of RANTES and MCP1 to hypothermia (7,53).

A few studies have examined cytokines in relation to MRI findings following HIE. Orrock et al. studied the association between serum cytokines measured at 24 and 72 hours and brain injury on MRI in 36 neonates treated with therapeutic hypothermia (53). Interleukin-6 and IL-10 were associated with death/abnormal MRI after controlling for baseline characteristics and severity of presentation. Massaro et al. studied the association between plasma samples collected at baseline and day 5 among 50 neonates treated with hypothermia and erythropoietin for NE(9). Elevated baseline IL1β, IL6, IL8, IL10, IL13, TNFα and IFNɣ were associated with increased severity of brain injury by MRI, but not one year outcome. When considered with the findings of Jenkins et al(7) and Chalak et al,(8) the relationship between IL-6, IL-8, IL10 and short term brain injury following HIE warrants further study. We found no association with 6–7 year outcomes.

The strengths of our study included the carefully defined inclusion criteria, the relatively large sample size given the rarity of the condition, and the detailed follow-up assessments performed by certified examiners who were blinded to cytokine and treatment data (assignment to hypothermia or usual care). Moreover, the assessment of neurodevelopmental outcomes at school age when functional assessments are deemed more accurate (as compared to 12–24 month assessments) is an advantage.

Important limitations included the lack of a healthy control group without encephalopathy, the measurement of cytokine/chemokine levels at only three time points, the absence of placental histology data, the use of blood spots stored over a prolonged period of time which may not accurately reflect biological activity, the multiple comparisons performed (raising the possibility of false positives) and our assumption that each incremental change in cytokine concentration may be associated with biological activity. Other investigators, evaluating cytokines data dichotomize cytokine concentrations as above and below the 75th percentile; this alternative approach assumes that high concentrations are more significant mediators of neurodevelopmental outcomes (54). For MCP-1 levels, some data exists to support a dose response pattern of risk. Circulating levels of MCP1 are associated with increased long-term stroke risk, with higher risk of ischemic stroke among individuals in the upper quartiles as compared with the first quartile (44).

Dried blood spot technology offers an important means for investigators to pool large data sets (even of entire populations); this methodology is employed in newborn screening programs throughout the world. It is not without inherent problems however. The biological stability of DBSS depends on multiple factors: temperature, humidity, transport, DBS/filter paper type, the particular biomarker evaluated, the specimen source and the assay performed (to name a few)(55). Optimal storage conditions for one biomarker may not be optimal for other analytes. With regards to the cytokines we studied, prior work by Skogstrand et al revealed that measurable amounts of most key cytokines were stable when stored for up to 23 years(12). However, decreased measured concentrations may be expected for IL-1, IL-8, sIL-6r, MMP-9, TREM1, CRP, BDNF and NT-4; the values and associations described for these analytes must be interpreted with caution.

CONCLUSION

Despite hypothermia to 33–35°C for 72 hours, 30% of infants with neonatal encephalopathy have poor outcomes (56, 57). Numerous adjuvant pharmacological agents are under investigation; however, a single drug or mechanistic target may fail to ameliorate brain injury among all patients. Biomarker discovery is an important area of research as measurement of perturbed cytokine-chemokine concentrations in the first week of life among neonates with encephalopathy has the potential to identify biological mechanisms that are disrupted and to disclose pathways that may be targeted to prevent poor outcomes. This work contributes two additional biomarkers for future testing.

Key Points.

  1. Elevation of specific cytokines and chemokines in neonates with encephalopathy has been noted along with increased risk of neurodevelopmental impairment in infancy. Data on impact of cytokines/chemokines on childhood outcome in neonatal encephalopathy is scarce.

  2. Cytokine/chemokines at <7 days were assessed among neonates in a trial of hypothermia for HIE in association with long term outcomes.

  3. Neonates who died or were impaired at 6–7 years following hypoxic-ischemic encephalopathy had lower RANTES and higher MCP-1 levels than those who survived without impairment.

Acknowledgments

The National Institutes of Health and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) provided grant support for the Neonatal Research Network’s Whole-Body Hypothermia Trial and its 6–7 Year School-age Follow-up through cooperative agreements. While NICHD staff did have input into the study design, conduct, analysis, and manuscript drafting, the content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Participating NRN sites collected data and transmitted it to RTI International, the data coordinating center (DCC) for the network, which stored, managed and analyzed the data for this study. On behalf of the NRN, Dr. Abhik Das (DCC Principal Investigator) and Mr. Scott A. McDonald (DCC Statistician) had full access to all of the data in the study, and with the NRN Center Principal Investigators, take responsibility for the integrity of the data and accuracy of the data analysis.

We are indebted to our medical and nursing colleagues and the infants and their parents who agreed to take part in this study. The following investigators, in addition to those listed as authors, participated in this study:

NRN Steering Committee Chairs: Alan H. Jobe, MD PhD, University of Cincinnati (2003–2006); Michael S. Caplan, MD, University of Chicago, Pritzker School of Medicine (2006–2011); Richard A. Polin, MD, Division of Neonatology, College of Physicians and Surgeons, Columbia University (2011-present).

Whole-body Hypothermia Subcommittee: Seetha Shankaran, MD, Chair (Wayne State University); Richard A. Ehrenkranz, MD, Vice Chair (Yale University); Ronald N. Goldberg, MD (Duke University); Abbot R. Laptook, MD (Brown University); Jon E. Tyson, MD, MPH (University of Texas Health Science Center at Houston); Michele C. Walsh, MD MS (Case Western Reserve University); Abhik Das, PhD (RTI International); Rosemary D. Higgins, MD (Eunice Kennedy Shriver National Institute of Child Health and Human Development); Ellen C. Hale, RN BS CCRC (Emory University); Karen J. Johnson, RN (University of Iowa).

Extended Hypothermia Subcommittee: Seetha Shankaran, MD, Chair (Wayne State University); Richard A. Ehrenkranz, MD, Vice Chair (Yale University); Susan R. Hintz, MD MS Epi (Stanford University); Athina Pappas, MD (Wayne State University); Jon E. Tyson, MD, MPH (University of Texas Health Science Center at Houston); Betty R. Vohr, MD (Brown University); Kimberly Yolton, PhD (Cincinnati Children’s Hospital Medical Center); Abhik Das, PhD (RTI International); Rosemary D. Higgins, MD (Eunice Kennedy Shriver National Institute of Child Health and Human Development); Rebecca Bara, RN BSN (Wayne State University).

Alpert Medical School of Brown University and Women & Infants Hospital of Rhode Island (U10 HD27904) – Betty R. Vohr, MD; William Oh, MD; Angelita M. Hensman, RN BSN; Bonnie E. Stephens, MD; Theresa M. Leach, MEd CAES; Lucy Noel; Victoria E. Watson, MS CAS.

Case Western Reserve University, Rainbow Babies & Children’s Hospital (U10 HD21364, M01 RR80) – Michele C. Walsh, MD MS; Avroy A. Fanaroff, MD; Deanne E. Wilson-Costello, MD; Nancy Bass, MD; Harriet G. Friedman MA; Nancy S. Newman, BA RN; Bonnie S. Siner, RN.

Cincinnati Children’s Hospital Medical Center and University of Cincinnati Medical Center (U10 HD27853, M01 RR8084) – Kurt Schibler, MD; Edward F. Donovan, MD; Kate Bridges, MD; Kimberly Yolton, PhD; Jean J. Steichen, MD; Barbara Alexander, RN; Cathy Grisby, BSN CCRC; Holly L. Mincey, RN BSN; Jody Hessling, RN; Teresa L. Gratton, PA.

Duke University School of Medicine, University Hospital, Alamance Regional Medical Center, and Durham Regional Hospital (U10 HD40492, M01 RR30) – Ronald N. Goldberg, MD; C. Michael Cotten, MD MHS; Kathryn E. Gustafson, PhD; Ricki F. Goldstein, MD; Kathy J. Auten, MSHS; Katherine A. Foy, RN; Kimberley A. Fisher, PhD FNP-BC IBCLC; Sandy Grimes, RN BSN; Melody B. Lohmeyer, RN MSN.

Emory University, Grady Memorial Hospital and Emory University Hospital Midtown (U10 HD27851, M01 RR39) – Barbara J. Stoll, MD; David P. Carlton, MD; Lucky Jain, MD; Ira Adams-Chapman, MD; Ann M. Blackwelder, RNC BS MS; Ellen C. Hale, RN BS CCRC; Sobha Fritz, PhD; Sheena Carter, PhD; Maureen Mulligan LaRossa, RN.

Eunice Kennedy Shriver National Institute of Child Health and Human Development – Linda L. Wright, MD; Elizabeth M. McClure, MEd; Stephanie Wilson Archer, MA.

Indiana University, University Hospital, Methodist Hospital, Riley Hospital for Children, and Wishard Health Services (U10 HD27856, M01 RR750) – Brenda B. Poindexter, MD MS; James A. Lemons, MD; Anna M. Dusick, MD FAAP (deceased); Diana D. Appel, RN BSN; Jessica Bissey, PsyD HSPP; Dianne E. Herron, RN; Lucy C. Miller, RN BSN CCRC; Leslie Richard, RN; Leslie Dawn Wilson, BSN CCRC.

McGovern Medical School at The University of Texas Health Science Center at Houston, Children’s Memorial Hermann Hospital, and Lyndon Baines Johnson General Hospital/Harris County Hospital District (U10 HD21373, M01 RR2588) – Kathleen A. Kennedy, MD MPH; Esther G. Akpa, RN BSN; Patty A. Cluff, RN; Patricia W. Evans, MD; Claudia I. Franco, RN BSN; Charles Green, PhD; Anna E. Lis, RN, BSN; Georgia E. McDavid, RN; Patti L. Pierce Tate, RCP; Nora I. Alaniz, BS; Pamela J. Bradt, MD MPH; Magda Cedillo; Susan Dieterich, PhD; Margarita Jiminez, MD; Terri Major-Kincade, MD MPH; Brenda H. Morris, MD; M. Layne Poundstone, RN BSN; Stacey Reddoch, BA; Saba Siddiki, MD; Maegan C. Simmons, RN; Laura L. Whitely, MD; Sharon L. Wright, MT; Lourdes M. Valdés PhD.

RTI International (U10 HD36790) – W. Kenneth Poole, PhD (deceased); Jane Hammond, PhD; Jeanette O’Donnell Auman, BS; Margaret Crawford, BS; Betty K. Hastings; Jamie E. Newman, PhD MPH; Carolyn M. Petrie Huitema, MS; Kristin M. Zaterka-Baxter, RN BSN.

Stanford University and Lucile Packard Children’s Hospital (U10 HD27880, M01 RR70) – Krisa P. Van Meurs, MD; David K. Stevenson, MD; Susan R. Hintz, MD MS Epi; Patrick D. Barnes, MD; M. Bethany Ball, BS CCRC; Maria Elena DeAnda, PhD; Barry E. Fleisher, MD; Anne M. DeBattista, RN PNP; Joan M. Baran, PhD; Julie C. Lee-Ancajas, PhD.

University of Alabama at Birmingham Health System and Children’s Hospital of Alabama (U10 HD34216, M01 RR32) – Namasivayam Ambalavanan, MD; Myriam Peralta-Carcelen, MD MPH; Kathleen G. Nelson, MD; Monica V. Collins, RN BSN MaEd; Shirley S. Cosby, RN BSN; Vivien A. Phillips, RN BSN; Laurie Lou Smith, EdS NCSP; ; Fred J. Biasini, PhD; Kirstin J. Bailey, PhD.

University of California-San Diego Medical Center and Sharp Mary Birch Hospital for Women (U10 HD40461) - Neil N. Finer, MD; David Kaegi, MD; Maynard R. Rasmussen, MD; Yvonne E. Vaucher, MD MPH; Martha G. Fuller, RN MSN; Radmila West PhD; Kathy Arnell, RNC; Chris Henderson, RCP CRTT; Wade Rich, BSHS RRT.

University of Rochester Medical Center, Golisano Children’s Hospital (U10 HD40521, M01 RR44) – Dale L. Phelps, MD; Gary J. Myers, MD; Diane Hust, MS RN CS; Linda J. Reubens, RN CCRC.

University of Miami Holtz Children’s Hospital (U10 HD21397, M01 RR16587) – Shahnaz Duara, MD; Charles R. Bauer, MD; Sylvia Hiriart-Fajardo, MD; Mary Allison, RN; Maria Calejo, MS;Ruth Everett-Thomas, RN MSN; Silvia M. Frade Eguaras, MA; Susan Gauthier, BA.

University of Texas Southwestern Medical Center at Dallas, Parkland Health & Hospital System, and Children’s Medical Center Dallas (U10 HD40689, M01 RR633) – Pablo J. Sánchez, MD; R. Sue Broyles, MD; Abbot R. Laptook, MD; Charles R. Rosenfeld, MD; Walid A. Salhab, MD; Roy J. Heyne, MD; Cathy Boatman, MS CIMI; Cristin Dooley, PhD LSSP; Gaynelle Hensley, RN; Jackie F. Hickman, RN; Melissa H. Leps, RN; Susie Madison, RN; Nancy A. Miller, RN; Janet S. Morgan, RN; Lizette E. Torres, RN; Alicia Guzman; Elizabeth Heyne, PA-C; Linda A. Madden, BSN RN CPNP; Sally Adams, PNP.

Wayne State University, Hutzel Women’s Hospital, and Children’s Hospital of Michigan (U10 HD21385) – Rebecca Bara, RN BSN; Yvette R. Johnson, MD MPH; Laura A. Goldston, MA; Geraldine Muran, RN BSN; Deborah Kennedy, RN BSN; Patrick J. Pruitt, BS.

Yale University, Yale-New Haven Children’s Hospital (U10 HD27871, M01 RR125, UL1 RR24139) – Richard A. Ehrenkranz, MD; Patricia Gettner, RN; Monica Konstantino, RN BSN; JoAnn Poulsen, RN; Elaine Romano, MSN; Joanne Williams, RN BSN; Susan DeLancy, MA CAS.

Source of funding

The National Institutes of Health and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) provided grant support for the Neonatal Research Network’s Whole-Body Hypothermia Trial and its 6–7 Year School-age Follow-up through cooperative agreements. This is a secondary study of ClinicalTrials.gov ID Whole-Body Cooling for Birth Asphyxia in Term Infants: NCT00005772.

Final Disclosures

Dr. Carlo reports having served on the advisory board of Pediatrix Medical Group and Paradigm Health and holding stock options at the Pediatrix Medical Group. Dr. Cotten reports having served on the data and safety monitoring board for the Inhibitex phase 3 study of Vernonate for the prevention of infections in preterm infants. Dr. Donovan reports having received support from the Environmental Protection Agency (Lanphear) and the Gerber Foundation. Dr. Stevenson reports having received research support from Pfizer. The other authors have no relevant financial relationships to disclose.

List of Abbreviations

CCL2

C-C motif chemokine ligand-2

CCL5

C-C motif chemokine ligand-5

CP

cerebral palsy

DBSS

dried blood spot samples

GMFCS

Gross Motor Function Classification System

HIE

hypoxic ischemic encephalopathy

IL

Interleukin

IQ

Intelligence quotient

LPS

lipopolysaccharide

MCP-1

monocyte chemotactic protein-1

MCP-2

monocyte chemotactic protein-2

MIP-1α

macrophage inflammatory protein-1α

MIP-1β

macrophage inflammatory protein-1β

MRI

magnetic resonance imaging

NE

neonatal encephalopathy

NICHD

Eunice Kennedy Shriver National Institute of Child Health and Human Development

NRN

Neonatal Research Network

NT-4

Neurotrophic factor-4

RANTES

Regulated upon Activation Normal T-cell Expressed and Secreted

TNF-α

Tumor necrosis factor-α

WPPSI-III

Wechsler Preschool and Primary Scale of Intelligence-III

WISC-IV

Wechsler Intelligence Scale for Children-IV

Footnotes

*

A complete list of group members appears in the Acknowledgements

Conflict of interest statement

There is no potential conflict of interest, real or perceived, with the study sponsor.

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