The effect of perinatal inflammation on neurodevelopmental

outcome in newborns at risk for hypoxic-ischemic encephalopathy

Research internship

Meike Jenster

S1805347

Supervisor Faculty Supervisor

H.C. Glass A. F. Bos

Department of Neurology

University of California, San Francisco

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Abstract Studies of preterm neonates suggest that infection may potentiate hypoxic-ischemic (HI) brain injury.

In term neonates, infection is a known risk factor for encephalopathy and cerebral palsy, however

whether it potentiates the risk of brain injury and adverse outcome in the setting of hypoxic-ischemic

encephalopathy (HIE) is not clear.

The charts of 257 term newborns with HIE were reviewed for signs of maternal and infant infection,

including chorioamnionitis and proven or suspected sepsis. Multivariate logistic regression was used

to assess the effect of infection on severity of brain injury as seen on a neonatal MRI (normal-mild vs.

moderate-severe), and on risk of adverse neurodevelopment at 30 months in a subset of subjects

(neuromotor score, NMS ≥2, or Bayley Scales of Infant Development II or III MDI <70 or cognitive

score <85).

Chorioamnionitis (42 subjects) was associated with a lower risk of moderate-severe brain injury (OR

0.3; 95%CI 0.1-0.7; p=0.003), and trended toward lower risk of adverse neurodevelopment. Infant

infection (32 subjects) trended toward association with moderate-severe injury (OR 1.6; 95%CI 0.8-

3.5; p=0.2), and was significantly associated with an abnormal NMS (OR 3.4; 95%CI 1.2-10.2;

p=0.03) but not cognitive outcome. After adjusting for hypothermia and severity of the HI insult,

maternal infection remained associated with a lower risk of brain injury, whereas the association

between infant infection and NMS was no longer significant.

These preliminary results are in keeping with animal studies that suggest that the timing of an

inflammatory signal may determine whether infection is injurious or protective.

Samenvatting Onderzoek in prematuur geboren kinderen heeft aangetoond dat infecties hypoxisch-ischemische (HI)

hersenschade kunnen potentieren. Infectie is een bekende risicofactor voor encefalopathie en cerebrale

parese in a terme neonaten. Het is echter nog niet bekend of infectie extra hersenschade veroorzaakt of

een negatieve invloed heeft op de ontwikkeling bij neonaten met hypoxisch-ischemische

encefalopathie (HIE).

Statussen van 257 a terme geboren neonaten met HIE werden onderzocht op de aanwezigheid van

maternale en neonatale infecties waaronder chorioamnionitis en aangetoonde of vermoedde sepsis.

Multivariabele regressie werd gebruikt om het effect van infectie op de ernst van de hersenschade,

gezien op een neonatale MRI scan (normaal-mild vs. matig-ernstig), en op het risico op een abnormale

ontwikkeling (gemeten na 30 maanden) te bepalen in een deel van het cohort. Hierbij werd een

neuromotore score (NMS) ≥2 en een Bayley Scales of Infant Development II of II MDI<70 of

cognitive score<85 als abnormaal gedefinieerd.

Chorioamnionitis (42 kinderen) was geassocieerd met een lager risico op matig-ernstige hersenschade

(OR 0.3; 95%CI 0.1-0.7; p=0.003) en neeg naar een associatie met een lager risico op abnormale

ontwikkeling. Neonatale infectie (32 kinderen) neeg naar een associatie met matig-ernstige

hersenschade (OR 1.6; 95%CI 0.8-3.5; p=0.2) en was significant geassocieerd met een abnormale

NMS (OR 3.4; 95%CI 1.2-10.2; p=0.03) maar niet met de cognitieve ontwikkeling. Na correctie voor

hypothermie en de ernst van het HI insult was maternale infectie nog steeds significant geassocieerd

met een verlaagd risico op hersenschade maar verdween de significante associatie tussen neonatale

infectie en de NMS.

Deze voorlopige resultaten komen overeen met wat dieronderzoek al eerder heeft aangetoond. Het

moment waarop het inflammatoire signaal optreedt kan bepalen of infectie schadelijk of juist

beschermend is.

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Table of Contents

1. Background

1.1 Definition Hypoxic-Ischemic Encephalopathy 5

1.2 Epidemiology 5

1.3 Etiology 5

1.4 Pathophysiology 6

1.5 Pattern of brain injury and the use of magnetic resonance imaging (MRI) 9

1.6 Neurodevelopmental outcome 10

1.7 Research question 10

2. Material and Methods 12

2.1 Patients 12

2.2 Clinical data 13

2.3 Magnetic resonance imaging 13

2.4 Neurodevelopmental Follow-Up 14

2.5 Statistics 14

3. Results 16

3.1 Patients 16

3.2 Brain injury on MRI 17

3.3 Neurodevelopmental outcome 17

4. Discussion and conclusions 19

5. References 22

6. Appendices 26

I The Sarnat Score 26

II The Encephalopathy Score 27

III Additional patient characteristics 28

IV Boxplot MRI scores with and without maternal inflammation 29

V Boxplot MRI scores with and without infant inflammation 29

VI Boxplot cognitive outcome with and without maternal inflammation 30

VII Boxplot cognitive outcome with and without infant inflammation 30

List of abbreviations

HI Hypoxia-Ischemia/Hypoxic-Ischemic

HIE Hypoxic-Ischemic Encephalopathy

ACOG American College of Obstetrics and Gynecology

NE Neonatal Encephalopathy

CBF Cerebral Blood Flow

BP Blood pressure

NO Nitric Oxide

TORCH-infections Toxoplasmosis, Other (syphilis), Rubella, Cytomegalovirus,

Herpes simplex virus

CP Cerebral Palsy

LPS Lipopolysaccharide

IL Interleukin

TNF Tumor Necrosis Factor

MRI Magnetic Resonance Imaging

OP Oligodendrocyte Progenitor

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W Watershed

BG/T Basal Ganglia/ Thalamus

PVL Periventricular Leukomalacia

DGN Deep Gray Nuclei

WMI White matter injury

GA Gestational Age

UA ph Umbilical cord artery pH

ES Encephalopathy Score

EEG Electroencephalography

WBC White blood cell count

ANC Absolute neutrophil count

I/T Immature to total neutrophil ratio

bpm beats per minute

SE Spin Echo

TR Repetition Time

TE Echo Time

DWI Diffusion Weighted Imaging

MDI Mental Development Index

BSID Bayley Scales of Infant Development

NMS Neuromotor score

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1. Background

1.1. Definition Hypoxic-Ischemic injury

Neonatal encephalopathy is a heterogeneous condition that presents in the first days of life,

characterized by damage of the central nervous system. If the underlying cause is perinatal

hypoxia-ischemia (HI), identified by clinical, laboratory or radiologic tests, the common term

is Hypoxic-Ischemic Encephalopathy (HIE). The American College of Obstetrics and

Gynecology (ACOG) determined four criteria that are required to define a perinatal HI event

sufficient to cause neurologic injury (Table 1)(1). However, researchers have not yet reached

consensus about the exact definition (2).

The clinical presentation of HIE varies according to the severity of brain injury. Infants with

severe HIE present with hypotonia, a depressed level of consciousness or coma, apnea, and

seizures. Scoring the clinical severity of encephalopathy can be useful to select infants that

require therapeutic intervention. The Sarnat Score(3) describes three different stages of

encephalopathy (mild, moderate, and severe) (Appendix 1), whereas the Encephalopathy

Score (4) is a scale from 0-6 (Appendix 2).

Criteria ACOG

Profound metabolic or mixed acidemia (pH < 7) in an umbilical artery blood sample, if

obtained

Persistence of an Apgar score of 0-3 for longer than 5 minutes

Neonatal neurologic sequelae (eg, seizures, coma, hypotonia)

Multiple organ involvement (eg, kidney, lungs, liver, heart, intestines) Table 1. Criteria defined by the American College of Obstetrics and Gynecology required to define a perinatal

HI event sufficient to cause neurologic injury

1.2 Epidemiology

HIE occurs in 2-8 per 1000 live full-term births in developed countries. The incidence

depends strongly on the used definition (2). In the United States the incidence is about 2 per

1000 live full-term births. (2). HIE is a major cause of mortality and later morbidity (5). Of

the children with moderate to severe HIE, 15-38% do not survive the newborn period, and

another 20-36% suffer from permanent neurologic damage (6-9). Infants with mild

encephalopathy tend to have a normal outcome (6,9,10). HIE occurs mainly in term infants.

Preterm infants can also suffer from HIE, but the underlying brain injury and clinical

manifestation is different (5).

1.3 Etiology

Hypoxia-ischemia is not always the cause of neonatal encephalopathy (NE). It accounts for

52% of all cases (11). In a large population-based control study, several antenatal risk factors

(e.g. maternal economic status, infertility treatment, maternal thyroid disease, severe

preeclampsia, post-dates, and intrauterine growth restriction), and intrapartum risk factors

(e.g. maternal fever, a persistant occipitoposterior position, and an acute intrapartum event)

have been associated with NE (12,13). Although these factors won’t cause HI injury

themselves, they may predispose HI events during labor through interference with normal

placental blood flow (14,15).

HI events can occur antepartum, intrapartum, and postnatal. The events that lead to HI insults

can have a maternal and/or placental origin (Table 2) (14). Timing of injury is difficult to

assess. In one study (11), antepartum causes (defined as dysgenetic and coagulation disorders)

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accounted for 13% of all cases. Fifty-six percent of all cases of newborn encephalopathy were

related to intrapartum events (involving HI, infections or intracranial haemorrhage). Two

percent of cases could be ascribed to a postnatal cause (adverse event within 7 days after

birth). In a study using magnetic resonance imaging (MRI) to assess timing of HI insults,

perinatally acquired brain injury was most common (16). However, this study did not exclude

the possibility that antenatal factors could initiate a causal pathway leading NE and that they

could make the neonatal brain more susceptible for HI injury.

Timing Cause of HI

Antepartum Maternal trauma

Impaired maternal oxygenation

- Anemia

- cardiopulmonary disease

Uterine hemorrhage

Intrapartum Inadequate perfusion maternal placenta (eg. abruption placentae)

Interruption of umbilical circulation (eg. Chord prolapse)

Uterine rupture

Prolonged/difficult labor

Postpartum Circulatory insufficiency

- recurrent apneic spells,

- large ductus arteriosus

- congenital heart disease

- pulmonary disease

vascular collapse (with sepsis)

Table 2. Events that cause hypoxia-ischemia by time of occurance.

Recently, several studies focused on maternal intrauterine infection and fetal systemic

inflammation as possible potentiators of perinatal HI brain injury. The results were

conflicting. Maternal fever was identified as an independent risk factor for term NE in two

studies (13,17). Chorioamnionitis was associated with an even higher risk for NE than

maternal fever alone (18). However, another research group found that histological

chorioamnionitis did not confer an additional risk for the development of HIE (19). The

different outcomes of both studies might be explained by the small sample size and the lack of

a control group in the last study.

1.4 Pathophysiology

1.4.1 Pathophysiology HIE

HI events in utero can cause profound brain damage. The severity and location of brain

damage depends on brain maturity, duration and severity of the insult(5,8,10). The complex

physiological and biochemical processes underlying HI brain injury are discussed in this

section.

Autoregulation

The unborn infant has defense mechanisms to deal with mild hypoxia and/or ischemia.

Reduction of uteroplacental blood flow, reduced normal respiration, or reduced oxygen

carrying capacity of the blood can lead to perinatal hypoxia. The brain cells of the fetus can

adapt to the hypoxic state through reduction of the energy consumption by suppressing

neuronal activity, and to switch to anaerobic metabolism (20). Ischemia occurs when cerebral

blood flow (CBF) decreases. Initially, compensatory mechanisms become activated to

maintain CBF during an asphyxial event. Due to the hypoxia and hypercapnia, peripheral

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vessels constrict, allowing more blood to flow to essential organs, including the brain. When

the first defenses fail because of profound hypoxia, CBF becomes dependent on blood

pressure (BP). When BP falls below autoregulatory threshold, CBF decreases (20,21). In

combination, the low brain oxygenation resulting from hypoxia, and low or absent cerebral

blood flow (CBF), lead to reduced glucose for metabolism and lactate acidosis (5,20).

Early and late onset injury

Neuronal HI injury is the result of a cascade of events following the decrease of glucose and

oxygen. Two phases of cell death have been identified.

First phase: oxidative stress and excitotoxic cell damage

Cell necrosis is the main cause of cell death in the first hours after the insult, but also

apoptosis plays a role (5,8). After the energy depletion, ATP-dependent Na+ /K

+ pumps fail,

causing cellular influx of sodium (Na+),

followed by chloride (Cl

-) and osmotic water influx .

This leads to cytotoxic edema and necrotic cell death (14). Furthermore, dysfunction of the

Na+ /K

+ pumps also results in failure of glutamate-reuptake, which is an important excitatory

neurotransmitter. Glutamate subsequently accumulates in the synaptic clefts and this leads to

over-activation of glutamate receptors. Induction of a cascade of events, involving

accumulation of calcium (Ca+), leads eventually to excitotoxic cell damage (figure 1) (5,14).

The accumulation of Ca+

in the cytoplasm causes production of nitric oxide (NO) and free

radicals, which can alter the cell membrane and lead to cell necrosis and apoptosis. The

neonatal brain is extremely susceptible for oxidative stress because of immature antioxidant

defenses.

Fig. 1. Relation between energy depletion and cell death. (from Volpe JJ. Neurology of the newborn. 5th ed.

Philadelphia: Saunders-Elsevier; 2008).

Second, late phase and the role of inflammation

The metabolic situation can stabilize after restoration of CBF, and oxygen and glucose

delivery(14,20,22). However, reperfusion can also cause additional injury in the late phase.

This phase starts 6-24 hours after the initial injury and its occurrence is dependent on duration

and severity of the HI insult, body temperature, gestational age, substrate availability and

preconditioning events (20,22). The cellular response after reperfusion is fairly similar to the

primary phase and is characterized by mitochondrial dysfunction, inflammation and apoptosis

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(5,14). Recently, late apoptosis was found to be important in the evolvement of HI injury, and

it may be more important than necrosis directly after injury (23).

The inflammatory response after HI is especially important in the context of infection.

Activation of the microglia (the brain’s phagocytes) in the first hours after HI results in the

release of free radicals and NO, as well as cytokines (Interleukin-1β (Il-1β) and tumor

necrosis factor-α (TNF-α)), which trigger inflammation at the site of injury (8). The role of

activated microglia in neuronal injury has been shown by the neuroprotective effect of

minocycline, an inhibitor of microglial activation, in neonatal rat (24).

1.4.2 Pathophysiology Infection in HIE

Infection/inflammation

Especially interesting is the synergetic effect of exposure of the fetus and neonate to infection

and hypoxia-ischemia. Several animal studies were conducted following epidemiological

evidence of a relation between maternal fever and chorioamnionitis and a worse

neurodevelopmental outcome in term newborns (25-28). Experimental animal studies showed

a potentiating effect of pretreatment with lipopolysaccharides (LPS), either intrauterine or

intraperitoneally, on HI brain injury (28-32). LPS is a molecule found on the outer membrane

of gram-negative bacteria, which are one of the most important pathogens in intrauterine

infection and neonatal sepsis. How systemic LPS makes its way through the blood-brain

barrier and causes brain damage is still unclear. However, once in the central nervous system,

LPS activates the Toll-like receptors 4 (TLR4) on microglia, which subsequently release the

cytokines Il-1β, Il-6 and TNF-α (33,34). The following inflammatory cascade is similar to the

inflammatory response seen after HI, but also after reperfusion and accumulation of

excitotoxins (14,35). It is unclear whether this inflammatory reaction is a direct cause of

neonatal brain injury, or whether it is a component in the cascade leading to brain injury after

an insult. Besides cytokine mediated brain injury, other mechanisms of LPS induced

inflammatory brain damage have been suggested. These include endothelial damage,

activation of pro-coagulant adhesion molecules, mitochondrial dysfunction from increased

NO production, and increased apoptosis (36).

Timing of LPS administration seems to be of great influence. Recently it was found that LPS

enhanced vulnerability of the neonatal brain if administered intraperitoneally either

immediately before (6 h or less) or more than 72 h before a period of 50 min of HI. When

administered in an intermediate period (24 h) before induction of HI, brain injury was

decreased(30). It appears that LPS can either enhance vulnerability of the developing brain to

HI, or protect the brain against HI, depending on the time of the infection. Whether this

variable effect of LPS on brain injury is also present in the human fetus, and whether the time

periods are the same, has yet to be elucidated.

The role of cytokines

The cytokines Il-1β, IL-6 and TNF-α, that are released by microglia in the inflammatory

response after HI and infection, have been associated with more extensive brain injury and

worse neurodevelopmental outcome in term newborns with NE (32,35,37,38). However, the

exact role of these inflammatory markers in HIE is still unclear. The potentiating effect is

thought to have two different origins: direct neurotoxicity and negative hemodynamic effects.

Firstly, cytokines are neurotoxic for oligodendrocyte progenitors (OPs). In term newborns,

50% of the oligodendrocyte population is the 04-positive immature oligodendrocyte. This cell

is very vulnerable for HI and infection/inflammation (39). Mature oligodendrocytes myelinate

the axons. Apoptosis of the OPs will therefore lead to a decrease in white matter. Secondly,

TNF-α could cause circulatory disturbances within vulnerable regions of the developing brain

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by various processes (e.g. systemic vasodilatation and hypoperfusion). These disturbances

may sensitize the brain, and may compromise the fetus so that even short periods of

hypoxia/ischemia could cause profound brain damage (10,12,20,25,36). It is unclear whether

this effect also applies to human pregnancies.

1.5 Patterns of brain injury and the use of magnetic resonance imaging

Patterns of brain injury after HIE

During brain development, different parts of the brain are vulnerable to HI brain damage,

depending on maturity and severity of the HI insult. In term newborns, certain neurons in the

deep gray nuclei and the perirolandic cortex are most likely to be damaged after asphyxia,

because of enhanced cytotoxic NO expression by neighboring cells. This leads to extra

oxidative stress and excitotoxicity (22,39). Preterm neonates mostly suffer from white

matter/oligodencrocyte injury, for OPs are especially vulnerable to oxidative stress and

glutamate, whereas mature oligodendrocytes are hightly resistant (8,40,41).

MRI scans could help determine the etiology and onset of brain injury. Two patterns of brain

injury have been identified in term NE: a watershed (W) predominant pattern, which involves

mostly white matter but, if extent, also cortical gray matter, and a basal ganglia/thalamus

(BG/T) predominant pattern involving the deep gray nuclei (DGN) and perirolandic cortex

(42). Term neonates typically present with damage in the DGN (43,44), although white matter

damage is also seen (10). The W area seems to be most likely affected after mild, prolonged

asphyxia and in infants with impaired autoregulation (14,45). The BG/T dominant pattern is

mostly seen after acute, severe asphyxia. Earlier, this study group found that the W pattern

was predominant in 45% of term newborns with HIE, in 25% the BG/T pattern was seen, and

30% had no brain damage (10).

Patterns of brain injury infection/inflammation and HIE

Animal studies have shown both white and gray matter injury in neonatal rats treated

intraperitoneally with LPS before induction of HI (28,29,46-49). However, in animals that

where LPS-exposed in utero, white matter injury was not present at all (31,32,50). This

suggests that timing of LPS administration could influence the pattern of injury. Neuronal

injury was seen in the cerebral neocortex, striatum, thalamus and hippocampus.

As stated before, microglia are thought to play a central role in the effect of perinatal infection

on brain damage. Some studies in preterm infants with periventricular leukomalacia (PVL)

focused on the role of microglia (24,39). Microglia cells are highly concentrated in the

cerebral white matter between 22 and 37 weeks, the time that hypoxia-ischemia and/or

infection are most likely to occur in preterm infants (51,52). After 37 weeks, their density in

the white matter decreases, and increases in the cerebral cortex, suggesting that the cerebral

cortex might be more damaged by the microglia by that time (51).

Although evidence from experimental animal studies shows a relation between perinatal

infection/inflammation and more severe brain injury in a HI model, this effect has not yet

been seen in clinical studies. One study group, focused on the role of cytokines Il-1, Il-6, Il-8

and TNF-α in term asphyxiated newborns, reported an association with lactate/choline

upregulation (indicating perturbation of cerebral oxidative metabolism) in the DGN, but not

in the W zone. However, blood level of cytokines did not differ between infants born to

mothers with clinical chorioamnionitis and those without chorioamnionitis (35). Total number

of mothers with clinical suspicion of chorioamnionitis was small (n=6). In contrast, another

study of a cohort of 61 neonates with a history of chorioamnionitis did find an association

between upregulation of cytokines and chorioamnionitis (53), so maternal inflammation

cannot be ruled out as an actor in the development of brain injury. The role of infant

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inflammation on brain injury in term newborns is unclear. However, in preterm infants, early

postnatal infection/inflammation was associated with an increased risk for white matter injury

(WMI) (54). Further clinical research is necessary to determine the effect of maternal and

neonatal infection/inflammation on pattern of brain injury in newborns with HIE.

1.6 Neurodevelopmental Outcome

Outcome after HIE

HIE is a major cause for mortality and later morbidity. Term newborns with HIE suffer from

a range of symptoms, depending on the severity of HIE. Cognitive deficits, CP, neurosensory

impairment and epilepsy are found in children with severe HIE(6,9,55).

Patterns of injury can be used to predict outcome after HIE. Both BG/T pattern and W pattern

have been associated with worse cognitive and motor outcomes, although not in the same

extend(10). Neonates suffering from HIE with BG/T predominant brain injury showed worse

cognitive and motor outcome, and more development of CP than those with the W dominant

pattern (16,56,57). Infants with isolated W injury only presented with cognitive impairment

(10,56,58). However, the BG/T predominant pattern was often accompanied by some W

injury and the more severe cognitive disabilities can therefore not be attributed to BG/T injury

alone.

Combined effect of infection/inflammation and HIE on outcome

Few studies have been done to examine the effect of maternal and infant inflammation on the

neurodevelopmental outcome, and the results were inconsistent. Studies focused on cytokines

and outcome showed an association between cytokines in HIE and abnormal

neurodevelopmental outcome at 12 (37) and 30 months (35). Other studies, focused more on

the direct role of infection on neurdevelopmental outcome, showed that clinical and

histological chorioamnionitis (25,27,59) and maternal fever (25) were associated with an

increased risk for CP. Furthermore, antenatal infection in combination with HIE conferred an

even higher risk for CP(25). These results highlight the possible potentiating effect of

infection on HI injury once more. However, no association was found between histological

chorioamnionitis and cognitive impairment(60). Furthermore, a maternal inflammatory state

was not associated with neurodevelopmental outcome, nor pattern of injury in another study

in newborns with HIE(10). However, these latter studies had limited criteria for perinatal

infection and sample size was small. Little is known about infant infection/inflammation and

outcome term newborns. A study in extremely premature infants demonstrated that proven

postnatal sepsis was associated with an adverse neurdevelopmental outcome, whereas

suspected sepsis was not(61). It is unclear whether this is also the case in term newborns with

HIE. More research is necessary to identify the effect of perinatal infection on

neurodevelopmental outcome.

1.7 Research question

Summary

Several reports have shown perinatal infection to be an independent risk factor for HIE.

Evidence from animal research suggests that perinatal infection and fetal systemic

inflammation are potentiators of perinatal HI brain injury. Inflammatory cytokines are thought

to play a key role in this potentiating effect, although the exact mechanism is not clear yet. An

association has been shown between upregulation of pro-inflammatory cytokines and

chorioamnionitis, pattern and extensiveness of brain injury, and neurodevelopmental outcome

in term newborns. Although the results from these studies are promising, a direct, additional

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effect of maternal and infant infection/inflammation on brain injury and outcome in term

newborns with HIE has not been shown yet.

Objective

The objective of this study was to determine the effect of perinatal inflammation on pattern

of brain injury, as assessed by early MRI, and to examine the effect of perinatal inflammation

on long term neurodevelopmental outcome in a cohort of term newborns with HIE.

Hypothesis

We hypothesized that maternal or infant inflammation in newborns with HIE would lead to

worse cognitive and motor outcome, and that it would cause more severe brain injury,

compared to neonates with HIE who did not show signs of infection/inflammation.

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2. Material and Methods

2.1 Patients

We included newborns derived from an ongoing prospective cohort study to the use of MRI

to predict outcome following HIE from 1993 till present (10,42,62-65). Neonates admitted to

the Intensive Care Nursery of the University of California, San Francisco were screened and

included in this cohort if they were ≥36 weeks gestational age (GA) by any measure (obstetric

dates, ultrasound, Ballard maturational age exam), and if any one of the following criteria was

present: a first blood gas or umbilical cord artery pH < 7.1, an umbilical cord artery or first

blood gas base deficit >10, a 5-minute Apgar score ≤ 5, and/or post asphyxic neurologic

syndrome that included stupor, diminished spontaneous movement, and hypotonia. These

inclusion criteria are broad, and were chosen to include newborns with a wide range in

severity of neonatal encephalopathy and neurodevelopmental outcome. Newborns with

suspected or confirmed congenital malformation, inborn error of metabolism or congenital

infection were excluded from the study. The University of California San Francisco’s

Committee on Human Research approved the research protocol. Infants were only included

after informed voluntary parental consent was obtained.

2.2 Clinical Data

Between December 1993 and May 2011, 309 newborns were enrolled in the cohort. One

newborn was excluded due to congenital malformation, and one due to inborn error of

metabolism. Twenty-six newborns were not studied, and were therefore excluded from this

study, leaving 282 newborns that met the inclusion criteria and underwent neonatal MRI.

Trained neonatal research nurses prospectively collected prenatal, perinatal and postnatal

variables from maternal and infant records. These variables included sex, birth weight,

gestational age (GA), APGAR score at 1, 5, and 10 minutes of life, delivery route, and

ethnicity. Furthermore, the amount of resuscitation was scored by using a resuscitation score:

1 = no intervention, 2 = blow-by oxygen, 3 = endotracheal suctioning, 4 = bag-mask positive

pressure ventilation, 5 = endotracheal intubation with positive pressure ventilation, and 6 =

endotracheal intubation with ventilation and medication (sodium bicarbonate with or without

epinephrine) (63). The degree of encephalopathy was measured in the first 3 days of life using

the encephalopathy score (ES), which ranges from 0 (no encephalopathy) to 6 (severe

encephalopathy) and is based on alertness, feeding, tone, respiratory status, reflexes, and

seizure activity (Appendix 2)(4).

Perinatal infection

The obstetric charts, neonatal charts and microbiology reports were retrospectively reviewed

for perinatal and postnatal infection. Signs and symptoms of maternal and fetal inflammation

and infection were defined as follows:

Maternal inflammation

1) Maternal fever was diagnosed if maternal axillary temperature was ≥ 37.8 °C within

72 hours of the delivery;

2) The history was positive for a prolonged rupture of membranes if membranes were

ruptured for ≥18 hours before delivery (66,67);

3) The presence of clinical chorioamnionitis was defined as maternal fever and uterine

tenderness, or as maternal fever or uterine tenderness and one of the following:

maternal tachycardia (>120 beats per minute (bpm)), fetal tachycardia (>160-180

bpm), purulent of foul-smelling amniotic fluid or vaginal discharge, maternal

leukocytosis (total blood leukocyte count > 15,000-18,000 cells/mm3) (68);

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4) Histological chorioamnonitis was diagnosed when placental pathology showed signs

of chorioamnionitis;

Infant inflammation

5) Bloodstream infection was diagnosed when blood cultures were positive for

pathogenic species within 7 days, other than staphylococcus epidermis (69);

6) Markers of culture negative clinical sepsis within 7 days: low (≤5,000 cells/mm3)

white blood cell count (WBC), low (<2,000 cells/mm3) absolute neutrophil count

(ANC), elevated (> 0.45) immature to total neutrophil ratio (I/T ratio), antibiotics ≥72

hours, and/or infant temperature ≥ 38.0°C(70-72). CRP levels were too inconsistent to

take into account.

None of the infants had meningitis.

Hereafter, maternal inflammatory status was defined as a positive history for clinical or

histological chorioamnionitis. Infant inflammation was made dichotomous as follows:

0 = no infant inflammation

1 = bloodstream infection or culture negative sepsis: if there was a low WBC (≤5,000

cells/mm3) or a low ANC (<2,000 cells/mm

3) or if there was a high I/T ratio (>

0.45) and an infant temperature >38.0°C

2.3 Magnetic Resonance Imaging

MRI protocol

MRI was performed in all newborns at a median of 5 days of life (range, 1-18 days). The aim

was to scan the newborns at 3 to 6 days of life, but only if they were stable for transport.

Transport to the MRI scanner was accompanied by a team of trained research nurses, and

newborns were moved in an MR compatible incubator (63,73). A specialized neonatal

circularly polarized head coil was used on a 1.5-Tesla Signa EchoSpeed system (GE Medical

Systems). Pentobarbital was used as sedation if necessary. Imaging sequences optimized for

the neonatal brain were used and included:

- T1-weighted sagittal and axial SE images with TR/TE of 500/11 ms, 4 mm thickness,

2 excitations and 192 × 256 acquisition matrix.

- T2-weighted axial dual echo, SE with TR/TE of 3000/60,120 ms, 4 mm thickness,1

excitation and 192 × 256 acquisition matrix (62).

- Diffusion weighted imaging (DWI) for subjects enrolled after the beginning of 1998,

SE echoplanar imaging diffusion sequence with TR/TE 7000/99 ms, field of view 180

mm, 3-mm thickness (no skip), 128 × 128 acquisition matrix, b value of 700 s/m2, six

directions (for some infants 30 directions were used), and three averages; some infants

had data obtained in 30 directions (63).

Scoring

T1-weighted images, T2-weighted images, and diffusion weighted images (for patients

enrolled after 1998) were scored prospectively by a pediatric neuroradiologist who was

blinded to the neonatal course. Injury to the BG/T and the W areas was scored independently

using a classification system that is predictive of neurodevelopmental outcome after neonatal

encephalopathy (Table 3) (42). After this evaluation, two additional outcome variables were

defined as previously described by this group (63): 1) Predominant pattern of injury, defined

as ‘basal nuclei-predominant’ (BG/T scores higher than W scores or maximum BG/T and W

scores), ‘Watershed-predominant’ (W scores higher than BG/T scores) or ‘normal’(BG/T en

W scores normal); and 2) severity of injury: normal-mild injury (BG/T score of 0 or 1 or W

score of 0,1 or 2) versus moderate-severe (BG/T score ≥ 2 or a W score of ≥ 3)(74).

14

Score Findings

Basal ganglia/thalamus

0 normal or isolated cortical infarct

1 abnormal signal in the thalamus

2 abnormal signal in the thalamus and lentiform nucleus

3 abnormal signal in the thalamus, lentiform nucleus, and

perirolandic cortex

4 more extensive involvement.

Watershed

0 Normal

1 single focal abnormality

2 abnormal signal in anterior or posterior watershed white

matter

3 abnormal signal in anterior or posterior watershed

cortex and white matter

4 abnormal signal in both anterior and posterior watershed

zones

5 more extensive cortical involvement. Table 3. MRI brain injury scoring system. Adapted from Barkovich, AJ et al. Prediction of neuromotor outcome

in perinatal asphyxia: Evaluation of MR scoring systems. Am J Neuroradiol 1998;19(1):143-149

2.4 Neurodevelopmental Follow-Up

Cognitive outcome was assessed at 30 months by a developmental psychologist who was

blinded to the neonatal course. Before June 2008 the Mental Development Index (MDI) of the

Bayley Scales of Infant Development II (BSID-II) was used (75,75). After June 2008

cognitive outcome was assessed by the Bayley Scales of Infant Development III (BSID-III)

(76). Both tests have mean scores of 100 with a SD of 15. Recently, several studies reported a

discrepancy between the outcomes of the cognitive/language scores of BSID-III and the MDI

of BSID-II. The BSID-III was significantly higher than the MDI of BSID-II (77-79).

Therefore, an MDI < 70 or mean cognitive and language score of <85 was classified as

abnormal.

At the same time point, a pediatric neurologist (who was also blinded to the imaging results

and clinical course) evaluated the neuromotor development by a validated scoring system

(neuromotor score (NMS)) based on tone, reflexes and power (80). In this score, normal is

scored as 0, abnormal tone or reflexes as 1, 2 is an abnormal tone and abnormal reflexes, 3 is

decreased power in addition to tone or reflex abnormality (functional deficit of power), if

there is involvement of cranial nerves with motor abnormality, the child gets a score of 4 and

if the child has a spastic quadraparesis, the score is 5. We classified a NMS of 0 or 1 as

normal, and NMS ≥ 2 as abnormal.

2.5 Statistics

Statistical analysis was performed using SPSS 16.0 (SPSS Inc, Chicago, Ill). Demographic,

clinical, and diagnostic characteristics were compared between newborns with and without

maternal or infant inflammation using χ2

and Fisher’s exact test for categorical variables,

Mann-Whitney U for non-parametric continuous variables, and Student’s T-test for normally

distributed continuous variables. Maternal and infant inflammatory status were compared

across the three patterns of injury using Fisher’s exact test. Logistic regression was used to

assess the association between the inflammatory status and severity of brain injury.

15

Hypothermia, GA and the umbilical cord artery pH (UA pH) were included in a multivariate

regression model. Univariate and multivariate logistic regression models (adjusting for

hypothermia and the UA pH) were also used to assess the association between inflammatory

status and abnormal cognitive and motor outcome. A p value of <0.05 was considered

significant.

16

3. Results

3.1 Patients

Table 4. Patient Characteristics by inflammatory status of 257 subjects at risk for hypoxic-ischemic brain injury.

Data are presented as number (%), Mean ± SD in case of normal distribution, and median (range) if the

distribution was skewed.

295 patients were enrolled during the study period. Of these, 3 subjects were excluded

because their MR images were not scored, 3 subjects were excluded because of missing

charts, and 32 were excluded because of insufficient documented information to assess

maternal inflammatory or infant inflammatory status, leaving 257 children that were included.

Placental pathology reports were available for 37 subjects, 33 from newborns born in UCSF,

2 from San Francisco General Hospital and 2 from Marin General Hospital. There were no

statistical differences in birth weight, GA, umbilical cord artery pH or base excess, or ES

between the included and excluded groups. However, APGAR scores at 5 minutes were

significantly higher in the excluded group. Furthermore, male sex, delivery route, death and

neonatal seizures on electroencephalography (EEG) were equally frequent in the excluded and

included subjects.

Forty-two (16%) newborns had a history of maternal inflammation, and 30 (12%) newborns

had a history of infant inflammation. Of the newborns with infant inflammation, 25 had

culture negative sepsis and 5 newborns had bloodstream infection. Newborns with a history of

maternal or infant inflammation had significantly higher first umbilical cord artery pH, lower

first umbilical cord base excess, and a higher resuscitation score. When comparing maternal

and infant inflammation separately with no inflammatory status, newborns with maternal

inflammation had significantly higher GA, cord artery pH, lower cord artery base excess and

higher resuscitation score. Newborns with infant inflammation had higher ES but other

clinical variables were not different (Appendix 3).

The 16 subjects that died had severe encephalopathy (13 (81%) newborns had the highest

ES), and more severe brain injury seen on MRI (50% had the maximum BG/T score, and 56%

had the maximum W score) compared to the survivors.

Patient Characteristics Maternal or infant

inflammation

No inflammation p

Total 66 191

Male sex 38 (57.6) 105 (55.0) 0.7

Caesarian section 41 (62.1) 98 (51.3) 0.1

Gestational Age, weeks 40.0 ± 1.6 39.5 ± 1.7 0.06

Birth weight, g 3268 ± 592 3388 ± 589 0.2

1-minute Apgar score 2 (0-7) 2 (0-8) 0.5

5-minute Apgar score 4 (0-9) 4 (0-9) 0.08

10-minute Apgar score 5 (0-10) 6 (0-9) 0.3

First umbilical cord artery

pH

base excess

7.1 ± 0.2

-10.5 ± 6.1

7.0 ± 0.2

-13.3 ± 7.0

0.01

0.01

Encephalopathy Score 4.5 (1-6) 4 (0-6) 0.6

Resuscitation Score 5 (3-6) 4 (1-6) 0.03

Neonatal Seizures on EEG 11 (16.7) 38 (19.9) 0.6

Hypothermia 23 (34.8) 65 (34.0) 0.9

Died 5 (7.6) 11 (5.8) 0.6

17

3.2 Perinatal infection and brain injury on MRI

Pattern of injury

In this cohort of newborns with HIE, brain injury seen on MRI was common. However, a

normal MRI scan was most frequent, seen in 100 (39%) newborns. The W predominant

pattern of injury was the most common pattern, seen in 98 (38%) newborns, and 59 (23%)

newborns had the BG/T predominant pattern. Newborns with maternal inflammation tended

to have a lower risk of injury in the BG/T area, but this difference was not significant (Table

5, p=0.05). Infant inflammation was significantly associated with pattern of injury. Newborns

with infant inflammation were more likely to have injury in the W pattern than to have a

normal MRI scan (53% vs. 19%, p=0.03).

Table 5. Inflammatory status across predominant pattern of brain injury. BG/T = Basal ganglia/ Thalamus.

Data are presented as number (% within inflammation status).

Severity of injury

Univariate analysis

Median (range) W score in the cohort was 1 (0-5) and median BG/T score was 0 (0-4).

Newborns with maternal inflammation tended to have lower W and BG/T scores but the

differences were not significant (p=0.07 and p=0.1 respectively) (Appendix 4). Newborns with

infant inflammation tended to have higher W scores but this difference was not significant

(p=0.07). BG/T scores were not significantly different between newborns with and without

infant inflammation (p=0.8) (Appendix 5). When considering the scores as a dichotomous

variable, moderate-severe brain injury was seen in 117 (46%) newborns, and 140 (54%)

newborns had normal to mild injury. Ten (24%) newborns with a history of maternal

inflammation had moderate-severe injury. Logistic regression showed that maternal

inflammation was significantly associated with a lower risk of moderate-severe brain injury

(OR 0.3; 95% CI 0.1-0.7; p=0.003). Of the newborns with infant inflammation, 17 (57%) had

moderate-severe injury. Infant inflammation tended to be associated with moderate-severe

injury but the association was not significant (OR 1.7; 95% CI 0.8-3.6; p=0.2).

Multivariable analysis

Adjusted for hypothermia, GA and the UA pH in a multivariable logistic model, maternal

inflammation was still associated with less severe brain damage (OR 0.3; 95% CI 0.1-0.8;

p=0.02). There was no significant association between infant inflammation and severity of

injury after correction for hypothermia and UA pH (OR 1.5; 95% CI 0.6-3.6; p=0.4).

3.3 Neurodevelopmental outcome

Sixteen newborns died before 30 months of age. Neither maternal inflammation (p=0.7), nor

infant inflammation (p=0.4) was associated with a higher risk of death after HIE. Motor

outcome was assessed in 126 (68%) surviving children that were old enough, and 106 (57%)

n (%) Total

N=257

Normal

N=100

Watershed

N=98

BG/T

N=59

P

Maternal

None 215 79 (36.7) 81 (37.7) 55 (25.6) 0.05

inflammation 42 21 (50.0) 17 (40.5) 4 (9.5)

Infant

None 225 94 (41.8) 81 (36.0) 50 (22.2) 0.03

inflammation 32 6 (18.8) 17 (53.1) 9 (28.1)

18

of the children had cognitive follow-up. Of the eligible infants that had no follow-up, 13 were

lost to follow-up, 38 missed the exam or were not studied, and 16 children were due for

examination at the time of writing. There were no significant differences in sex, delivery

route, neonatal seizures on EEG, birth weight, GA, ES, APGAR scores, or presence of

inflammation between the children with and without follow-up.

Motor outcome

The median (range) NMS was 1 (0-6) for children with and also 1 (0-6) for children without

maternal inflammation. The median NMS was 2 (0-6) for children with and 1 (0-6) for

children without infant inflammation. In this cohort, 46 (23%) children had an abnormal NMS

(NMS ≥2). Of the children with a history of maternal inflammation, 4 (22%) had an abnormal

NMS. In univariate logistic regression analysis, maternal inflammation was not significantly

associated with an abnormal NMS (table 6. p=0.2). Ten (63%) of the children with infant

inflammation had an abnormal NMS. Infant inflammation was associated with an abnormal

NMS in univariate logistic regression (table 6. P=0.03). After adjusting for hypothermia and

the UA pH, this association was no longer significant (OR 2.6; 95% CI 0.7-10.0; p=0.2).

Cognitive outcome

In total, 75 children were tested with the BSID-II with a median (range) MDI of 86 (<50-

121). After 2008, the cognitive outcome was assessed with BSID-III in 31 children. Median

cognitive/language composite score was 102 (71-121). Considering both scores, 21 (20%) of

the assessed children had an abnormal cognitive outcome (MDI <70 or cognitive score <85).

None of the children with a history of maternal inflammation had an abnormal cognitive

outcome so logistic regression was not possible. Maternal inflammation was associated with

an abnormal cognitive outcome when using χ2 (p=0.04). Four (33%) of the children with

infant inflammation had an abnormal cognitive outcome. In both univariate and multivariate

analyses, infant inflammation trended toward association with a higher risk for an abnormal

cognitive outcome but in both analyses the association was not significant (table 6).

Table 6. Logistic regression: Odds ratios (OR) and 95% Confidence Intervals (CI) of maternal and infant

inflammation for NMS ≥ 2 and abnormal cognitive score at 30 months Univariate analyses show the unadjusted

relationship between inflammation and outcome, while in multivariate analyses there was adjusted for

hypothermia and the umbilical cord artery pH.

Univariate analyses Multivariate analyses

OR 95% CI P OR 95% CI P

NMS≥2

Maternal inflammation 0.4 0.1-1.5 0.2 0.4 0.09-1.4 0.1

Infant inflammation 3.4 1.2-10.2 0.03 2.6 0.7-10.0 0.2

MDI < 70 or cog < 85

Maternal inflammation - - - - - -

Infant inflammation 2.3 0.6-8.4 0.2 3.6 0.7-18.3 0.1

19

4. Discussion In this cohort of newborns at risk for HIE, maternal inflammation (histological or clinical

chorioamnionitis) was associated with a lower risk for brain injury, whereas infant

inflammation (culture negative sepsis or bloodstream infection) trended toward association

with moderate-severe injury. Follow-up at 30 months showed no association between

maternal inflammation and outcome. Infant inflammation was associated with motor

impairment at 30 months, though this association was not independent from the severity of

the perinatal insult. The variable effect of inflammation on brain injury is in keeping with animal studies that

suggest that the timing of an inflammatory signal may determine whether inflammation is

injurious or protective (29,30). Induction of inflammation with intraperitoneal administration

of LPS, either 4-6 hours, or more than 72 hours before HI had a potentiating effect, whereas

LPS administration 24 hours before HI had a preconditioning effect. The exact mechanisms

remain unclear, but the protecting effect is thought to be attributable to an upregulation of

TNF-α, TGF-1β, antioxidants, and corticosteroids, which can be neuroprotective (28,29,48).

This present study might suggest that timing of the inflammatory signal also influences

whether infection is harmful or protective in term newborns. However, it is difficult to assess

the exact timing of an inflammatory signal. Although postnatal infection/inflammation

usually occurs later in time than chorioamnionitis, infant inflammation can also be an effect

of intrauterine infection, and the exact moment of initiation of the complex interaction with

HI is therefore not clear. In this cohort, histological or clinical chorioamnionitis was associated with a lower risk for

brain injury, mainly for injury in the BG/T pattern. This result is in keeping with an earlier

study in this same cohort that showed that maternal inflammatory state (clinical

chorioamnionitis or maternal fever) was associated with lower BG/T scores after adjusting for

maternal substance use, prolonged rupture of membranes and birth weight (10). However, this

is not supported by animal studies that reported enhancement of brain injury, mainly in the

grey matter, after intrauterine exposure to LPS (31,32). In human newborns, there is few

published research to the direct effect of chorioamnionitis to brain injury seen on MRI.

However, evidence of the role of cytokines and chorioamnionitis in the development of brain

injury is conflicting. Upregulation of the inflammatory cytokines IL-6 and IL-8, which have

been linked to brain injury after HI (35,37,38), was associated with chorioamnionitis by

Shalak et al.(53), but not in another study of this cohort by Bartha et al.(35). More research to

the effect of other, possible neuroprotective, cytokines is necessary to clarify the underlying

pathophysiology of the relation between chorioamnionitis and brain injury.

The finding that infant inflammation was associated with the W pattern of injury is in keeping

with earlier research in animals (29,50), and with a study in preterm infants that reported an

association between postnatal sepsis and WMI (54). Systemic inflammation in newborns is

thought to disturb cerebral autoregulation and could therefore make the brain more

susceptible for HI. Because of the prolonged nature of the HI after inflammation (rather than

an acute event) it is more likely that the W area is injured than the grey matter, as injury in the

W area has been associated with prolonged mild HI (14). Another explanation for the

predominance of the W pattern is that a part of the population of oligodendrocytes is still

immature in term newborns. These oligodendrocytes are very vulnerable for HI. Although

most previous studies of infant inflammation and brain injury were focused on preterm

20

neonates, our study suggests that term neonates might also be vulnerable to WMI after infant

inflammation and HIE. However, infant inflammation was not significantly associated with

the severity of brain injury. Although infant inflammation tended towards association in

univariate analysis, there was no association after adjusting for hypothermia and the umbilical

cord artery pH. This suggests that infant inflammation confers a risk for more injury in the W

area, but that this injury is mostly mild and dependent on the severity of the ischemia and

hypothermia. Also, early MRI might not be sensitive enough to detect mild injury (35). Interestingly, there was no independent association with either maternal or infant

inflammation on neurodevelopmental outcome in this cohort. Many studies have investigated

the relation between chorioamnionitis and CP in preterm infants, but only one study showed

an association between clinical chorioamnionitis and CP in term newborns (25). In contrast,

this study showed that maternal inflammation trended toward association with lower NMS in

both univariate and multivariate analyses, but the difference was not significant. This might

suggest that the reduction of brain injury seen on early MRI was not enough to reduce the risk

of an abnormal motor outcome. Furthermore, none of the children with maternal

inflammation had an abnormal cognitive outcome. However, based on the small numbers, it

is not possible draw conclusions on the effect of maternal inflammation on cognitive

outcome. The association between infant inflammation and a worse motor outcome could be a

consequence of the early brain injury, seen on the MRI. However, the association with both

brain injury and the worse motor outcome was no longer present after correction for the

umbilical cord artery pH. This suggests that this association is dependent on the severity of

the HI insult. Determining the independent effect of infection is difficult. The underlying inflammatory

pathway is similar to the one seen after HI and seizures. To identify the effect of

inflammation, independent of the severity of the perinatal HI, we adjusted for the umbilical

cord artery pH in a multivariable model. However, UA pH alone might not be a sufficient

predictor for the severity of the HI insult. Still, we did not adjust for the ES, for it is difficult

to measure whether the neonates are encephalopathic due to infection/inflammation or HI. As

animal studies suggest that inflammation can potentiate brain injury, even when the hypoxia-

ischemia itself is not severe enough to cause injury, it is possible that the clinical presentation

of encephalopathy in the first days of life could reflect injury caused by inflammation. Also,

newborns with infant inflammation had higher ES scores then the ones without in this cohort.

Therefore, adjusting for the ES in multivariate analyses might not be appropriate. In order to

determine the separate effects of HI and infection/inflammation, more elaborate and specific

criteria for both predictors are necessary. We acknowledge the limitations of this study. First, it is difficult measure maternal and infant

inflammation since both conditions have a wide variability in presentation and severity.

Differences in quality and detail of the clinical reports, especially from children born in the

beginning of the cohort, made it difficult to assess clinical chorioamnionitis. Second, despite the large size of the total cohort, we had small numbers of the predictors and

outcome of interest. Therefore, our conclusions should be read with caution. Also, because of unavailability of a blood count for a part of the cohort, we excluded the

children without a blood count. However, we thereby might have excluded children with less

severe injury, as a blood draw was less urgent for these children in the newborn period.

21

Furthermore, part of the cohort was lost to follow-up at 30 months. It appears that parents that

are most concerned, are unwilling to come to clinic. Hereby, we might have missed more

severely damaged children. Finally, we did not account for the possible confounding effect of

socioeconomic background of the primary caregiver on neurodevelopmental outcome (81).

This might have obscured our ability to see an independent effect of maternal or infant

inflammation on outcome at 30 months.

Conclusion These preliminary results suggest that the timing of an inflammatory signal may determine

whether infection is injurious or protective. In this study, maternal inflammation seemed to

have a protective effect on brain injury after HI, whereas infant inflammation appeared to be

more injurious.

In order to determine the exact influence of timing of infection in relation to HI, more basic

research is necessary to elucidate the underlying pathophysiologic mechanisms. Also,

measuring cytokines might help clarifying the relation between brain injury and inflammation

after maternal or infant infection and HI. Despite the preliminary nature of these results,

newborns with HIE and suspected sepsis should be treated with caution.

22

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6. Appendices

Appendix I

The Sarnat score

Appendix 1. The Sarnat score. Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A

clinical and electroencephalographic study. Arch Neurol 1976 Oct;33(10):696-705.

27

Appendix II

The Encephalopathy Score

Encephalopathy sign Score = 0 Score = 1

Feeding Normal Gavage feeds, gastrostomy

tube or not tolerating oral

feeds

Alertness Alert Irritable, poorly responsive

or comatose

Tone Normal Hypotonia or hypertonia

Respiratory status Normal Respiratory distress (need for

CPAP or mechanical

ventilation)

Reflexes Normal Hyperreflexia, hyporeflexia

or absent reflexes

Seizure None Suspected or confirmed

clinical seizure

Total 0-6 Appendix 2. The Encephalopathy Score. Newborn infants were scored daily for the first 3 days of life and the

maximum score was used for analysis. The ES was assigned only on days the subject was not sedated or

paralyzed. Miller SP, Latal B, Clark H, Barnwell A, Glidden D, Barkovich AJ, et al. Clinical signs predict 30-

month neurodevelopmental outcome after neonatal encephalopathy. Obstet Gynecol 2004;190(1):93-99.

28

Appendix III

Appendix 3. Patient Characteristics by maternal and infant inflammatory status of 257 subjects at risk for

hypoxic-ischemic brain injury. Data are presented as number (%), Mean ± SD in case of normal distribution and

median (range) if the distribution was skewed.

Patient Characteristics Maternal

Inflammation

No infl p Infant

inflammation

No infl p

Total 42 215 30 227

Male sex 26 (61.9) 117 (54.4) 0.4 15 (50.0) 128 (56.4) 0.5

Caesarian section 22 (60.0) 113 (52.6) 0.3 20 (66.7) 119 (52.4) 0.2

Gestational Age, weeks 40.2 ± 1.3 39.5 ± 1.7 0.002 39.7 ± 2.0 39.6 ± 1.6 0.8

Birth weight, g 3307 ± 522 3365 ± 604 0.6 3343 ± 702 3370 ±

575 0.3

1-minute Apgar score 2 (0-7) 2 (0-8) 0.4 2 (0-6) 2 (0-8) 0.9

5-minute Apgar score 4 (0-9) 4 (0-9) 0.1 4 (0-9) 4 (0-9) 0.7

10-minute Apgar score 5 (0-10) 6 (0-9) 0.3 5 (2-9) 5 (0-10) 0.7

First umbilical cord artery

pH

base excess

7.1 ± 0.2

-8.3 ± 4.7

7.0 ± 0.2

-13.5 ± 7.0

0.001

0.000

7.0 ± 0.2

-14.0 ± 6.3

7.0 ± 0.2

-12.5 ±

7.0

0.9

0.4

Encephalopathy Score 4 (1-6) 5 (0-6) 0.1 5.5 (1-6) 4 (0-6) 0.02

Resuscitation Score 5 (3-6) 4 (1-6) 0.006 4.5 (4-6) 5 (1-6) 1.0

Neonatal Seizures on EEG 8 (19.0) 41 (19.1) 1.0 4 (13.3) 45 (19.8) 0.4

Hypothermia 18 (42.5) 70 (32.6) 0.2 8 (26.7) 80 (35.2) 0.4

Died 2 (4.8) 14 (6.5) 0.7 3 (10.0) 13 (5.7) 0.4

29

Appendix IV

Boxplot Watershed and basal ganglia/thalamus scores with and without maternal inflammation

P=0.07 P=0.1 Appendix 4. p values calculated with Mann-Whitney U test.

Appendix V

Boxplot Watershed and basal ganglia/thalamus scores with and without infant inflammation

P=0.07 P =0.8 Appendix 4. p values calculated with the Mann-Whitney U test.

30

Appendix VI

Boxplot of cognitive outcome of children with and without maternal inflammation

P=0.4 P=0.4 P=0.7 Appendix 6. P values calculated with the Mann-Whitney U test.

Appendix VII

Boxplot of cognitive outcome of children with and without infant inflammation

P=0.4 P=0.8 P=0.1 Appendix 7. P values calculated with the Mann-Whitney U test.