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
2
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.
3
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
4
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
5
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)
6
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
7
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
8
(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
9
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
10
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
11
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.
12
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);
13
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
5. References
(1) American College of Obstetricians and Gynecologist and American Academy of Pediatrics. Background
Neonatal Encephalopathy and Cerebral Palsy: Defining the Pathogenesis and Pathophysiology. Washington,
D.C: American College of Obstetricians and Gynecologist Distribution Center; 2003.
(2) Graham EM, Ruis KA, Hartman AL, Northington FJ, Fox HE. A systematic review of the role of intrapartum
hypoxia-ischemia in the causation of neonatal encephalopathy. Am J Obstet Gynecol 2008 Dec;199(6):587-595.
(3) Sarnat HB, Sarnat MS. Neonatal encephalopathy following fetal distress. A clinical and
electroencephalographic study. Arch Neurol 1976 Oct;33(10):696-705.
(4) 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.
(5) Volpe JJ. Perinatal brain injury: from pathogenesis to neuroprotection. Ment Retard Dev Disabil Res Rev
2001;7(1):56-64.
(6) Perlman M, Shah PS. Hypoxic-ischemic encephalopathy: challenges in outcome and prediction. J Pediatr
2011 Feb;158(2 Suppl):e51-4.
(7) Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA, Donovan EF, et al. Whole-body
hypothermia for neonates with hypoxic-ischemic encephalopathy. N Engl J Med 2005 Oct 13;353(15):1574-
1584.
(8) Ferriero DM. Medical progress: Neonatal brain injury. N Engl J Med 2004;351(19):1985-1995.
(9) Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, Ferriero DM, et al. Selective head cooling
with mild systemic hypothermia after neonatal encephalopathy: multicentre randomised trial. The Lancet 2005
2/25;365(9460):663-670.
(10) Miller SP, Ramaswamy V, Michelson D, Barkovich AJ, Holshouser B, Wycliffe N, et al. Patterns of brain
injury in term neonatal encephalopathy. J Pediatr 2005;146(4):453-460.
(11) Pierrat V, Haouari N, Liska A, Thomas D, Subtil D, Truffert P. Prevalence, causes, and outcome at 2 years
of age of newborn encephalopathy: Population based study. Archives of Disease in Childhood: Fetal and
Neonatal Edition 2005;90(3):F257-F261.
(12) Badawi N, Kurinczuk JJ, Keogh JM, Alessandri LM, O'Sullivan F, Burton PR, et al. Antepartum risk
factors for newborn encephalopathy: the Western Australian case-control study. BMJ 1998 Dec
5;317(7172):1549-1553.
(13) Badawi N, Kurinczuk JJ, Keogh JM, Alessandri LM, O'Sullivan F, Burton PR, et al. Intrapartum risk factors
for newborn encephalopathy: The Western Australian case-control study. Br Med J 1998;317(7172):1554-1558.
(14) Volpe JJ. Neurology of the newborn. 5th ed. Philadelphia: Saunders-Elsevier; 2008.
(15) Hagberg B, Hagberg G, Beckung E, Uvebrant P. Changing panorama of cerebral palsy in Sweden. VIII.
Prevalence and origin in the birth year period 1991-94. Acta Paediatrica, International Journal of Paediatrics
2001;90(3):271-277.
(16) Cowan F, Rutherford M, Groenendaal F, Eken P, Mercuri E, Bydder GM, et al. Origin and timing of brain
lesions in term infants with neonatal encephalopathy. Lancet 2003;361(9359):736-742.
(17) Impey LWM, Greenwood CEL, Black RS, Yeh PS-, Sheil O, Doyle P. The relationship between
intrapartum maternal fever and neonatal acidosis as risk factors for neonatal encephalopathy. Obstet Gynecol
2008;198(1):49.e1-49.e6.
(18) Blume HK, Li CI, Loch CM, Koepsell TD. Intrapartum fever and chorioamnionitis as risks for
encephalopathy in term newborns: A case-control study. Dev Med Child Neurol 2008;50(1):19-24.
(19) Shalak L, Johnson-Welch S, Perlman JM. Chorioamnionitis and Neonatal Encephalopathy in Term Infants
With Fetal Acidemia: Histopathologic Correlations. Pediatr Neurol 2005 9;33(3):162-165.
(20) Gunn AJ, Bennet L. Fetal hypoxia insults and patterns of brain injury: insights from animal models. Clin
Perinatol 2009 Sep;36(3):579-593.
(21) Papile LA, Rudolph AM, Heymann MA. Autoregulation of cerebral blood flow in the preterm fetal lamb.
Pediatr Res 1985 Feb;19(2):159-161.
(22) Wachtel EV, Hendricks-Muñoz KD. Current Management of the Infant Who Presents with Neonatal
Encephalopathy. Current Problems in Pediatric and Adolescent Health Care 2011 6;41(5):132-153.
(23) Hu BR, Liu CL, Ouyang Y, Blomgren K, Siesjo BK. Involvement of caspase-3 in cell death after hypoxia-
ischemia declines during brain maturation. J Cereb Blood Flow Metab 2000 Sep;20(9):1294-1300.
(24) Fan L-, Pang Y, Lin S, Tien L-, Ma T, Rhodes PG, et al. Minocycline reduces lipopolysaccharide-induced
neurological dysfunction and brain injury in the neonatal rat. J Neurosci Res 2005;82(1):71-82.
(25) Grether JK, Nelson KB. Maternal infection and cerebral palsy in infants of normal birth weight. JAMA
1997 Jul 16;278(3):207-211.
23
(26) Wu YW. Systematic review of chorioamnionitis and cerebral palsy. Ment Retard Dev Disabil Res Rev
2002;8(1):25-29.
(27) Wu YW, Colford JM. Chorioamnionitis as a risk factor for cerebral palsy: A meta-analysis. J Am Med
Assoc 2000;284(11):1417-1424.
(28) Ikeda T, Mishima K, Aoo N, Egashira N, Iwasaki K, Fujiwara M, et al. Combination treatment of neonatal
rats with hypoxia-ischemia and endotoxin induces long-lasting memory and learning impairment that is
associated with extended cerebral damage. Obstet Gynecol 2004;191(6):2132-2141.
(29) Eklind S, Mallard C, Leverin A-, Gilland E, Blomgren K, Mattsby-Baltzer I, et al. Bacterial endotoxin
sensitizes the immature brain to hypoxic-ischaemic injury. Eur J Neurosci 2001;13(6):1101-1106.
(30) Eklind S, Mallard C, Arvidsson P, Hagberg H. Lipopolysaccharide induces both a primary and a secondary
phase of sensitization in the developing rat brain. Pediatr Res 2005;58(1):112-116.
(31) Larouche A, Roy M, Kadhim H, Tsanaclis AM, Fortin D, Sébire G. Neuronal injuries induced by perinatal
hypoxic-ischemic insults are potentiated by prenatal exposure to lipopolysaccharide: Animal model for
perinatally acquired encephalopathy. Dev Neurosci 2005;27(2-4):134-142.
(32) Burd I, Brown A, Gonzalez JM, Chai J, Elovitz MA. A mouse model of term chorioamnionitis: Unraveling
causes of adverse neurological outcomes. Reproductive Sciences 2011;18(9):900-907.
(33) Kopp EB, Medzhitov R. The Toll-receptor family and control of innate immunity. Curr Opin Immunol
1999;11(1):13-18.
(34) Hagberg H, Gilland E, Bona E, Hanson L-, Hahn-Zoric M, Blennow M, et al. Enhanced expression of
interleukin (IL)-1 and IL-6 messenger RNA and bioactive protein after hypoxia-ischemia in neonatal rats.
Pediatr Res 1996;40(4):603-609.
(35) Bartha AI, Foster-Barber A, Miller SP, Vigneron DB, Glidden DV, Barkovich AJ, et al. Neonatal
encephalopathy: Association of cytokines with MR spectroscopy and outcome. Pediatr Res 2004;56(6):960-966.
(36) Ugwumadu A. Infection and fetal neurologic injury. Curr Opin Obstet Gynecol 2006 Apr;18(2):106-111.
(37) Aly H, Khashaba MT, El-Ayouty M, El-Sayed O, Hasanein BM. IL-1β, IL-6 and TNF-α and outcomes of
neonatal hypoxic ischemic encephalopathy. Brain and Development 2006;28(3):178-182.
(38) Martín-Ancel A, García-Alix A, Pascual-Salcedo D, Cabañas F, Valcarce M, Quero J. Interleukin-6 in the
cerebrospinal fluid after perinatal asphyxia is related to early and late neurological manifestations. Pediatrics
1997;100(5):789-794.
(39) Volpe JJ, Kinney HC, Jensen FE, Rosenberg PA. Reprint of "The developing oligodendrocyte: Key cellular
target in brain injury in the premature infant". International Journal of Developmental Neuroscience
2011;29(6):565-582.
(40) Jensen FE. Role of glutamate receptors in periventricular leukomalacia. J Child Neurol 2005
Dec;20(12):950-959.
(41) Rees S, Harding R, Walker D. The biological basis of injury and neuroprotection in the fetal and neonatal
brain. Int J Dev Neurosci 2011 Oct;29(6):551-563.
(42) Barkovich AJ, Hajnal BL, Vigneron D, Sola A, Partridge JC, Allen F, et al. Prediction of neuromotor
outcome in perinatal asphyxia: Evaluation of MR scoring systems. Am J Neuroradiol 1998;19(1):143-149.
(43) Okereafor A, Allsop J, Counsell SJ, Fitzpatrick J, Azzopardi D, Rutherford MA, et al. Patterns of brain
injury in neonates exposed to perinatal sentinel events. Pediatrics 2008;121(5):906-914.
(44) McQuillen PS, Ferriero DM. Selective vulnerability in the developing central nervous system. Pediatr
Neurol 2004 4;30(4):227-235.
(45) Martin E, Barkovich AJ. Magnetic resonance imaging in perinatal asphyxia. Arch Dis Child 1995;72(1
SUPPL.):F62-F70.
(46) Lehnardt S, Massillon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, et al. Activation of innate immunity
in the CNS triggers neurodegeneration through a Toll-like receptor 4-dependent pathway. Proc Natl Acad Sci U
S A 2003;100(14):8514-8519.
(47) Yang L, Sameshima H, Ikeda T, Ikenoue T. Lipopolysaccharide administration enhances hypoxic-ischemic
brain damage in newborn rats. J Obstet Gynaecol Res 2004;30(2):142-147.
(48) Wang X, Hagberg H, Nie C, Zhu C, Ikeda T, Mallard C. Dual role of intrauterine immune challenge on
neonatal and adult brain vulnerability to hypoxia-ischemia. J Neuropathol Exp Neurol 2007;66(6):552-561.
(49) Wang X, Hagberg H, Zhu C, Jacobsson B, Mallard C. Effects of intrauterine inflammation on the
developing mouse brain. Brain Res 2007;1144(1):180-185.
(50) Coumans ABC, Middelanis J, Garnier Y, Vaihinger H-, Leib SL, Von Duering MU, et al. Intracisternal
application of endotoxin enhances the susceptibility to subsequent hypoxic-ischemic brain damage in neonatal
rats. Pediatr Res 2003;53(5):770-775.
(51) Monier A, Adle-Biassette H, Delezoide A-, Evrard P, Gressens P, Verney C. Entry and distribution of
microglial cells in human embryonic and fetal cerebral cortex. J Neuropathol Exp Neurol 2007;66(5):372-382.
24
(52) Rezaie P, Male D. Colonisation of the developing human brain and spinal cord by microglia: A review.
Microsc Res Tech 1999;45(6):359-382.
(53) Shalak LF, Laptook AR, Jafri HS, Ramilo O, Perlman JM. Clinical chorioamnionitis, elevated cytokines,
and brain injury in term infants. Pediatrics 2002;110(4):673-680.
(54) Chau V, Poskitt KJ, McFadden DE, Bowen-Roberts T, Synnes A, Brant R, et al. Effect of chorioamnionitis
on brain development and injury in premature newborns. Ann Neurol 2009;66(2):155-164.
(55) Marcdante K, Kliegman R, Jenson H, Behrman R. Nelson Essentials of Pediatrics. 6th ed. Philadelphia:
Saunders; 2011.
(56) Martinez-Biarge M, Diez-Sebastian J, Kapellou O, Gindner D, Allsop JM, Rutherford MA, et al. Predicting
motor outcome and death in term hypoxic-ischemic encephalopathy. Neurology 2011;76(24):2055-2061.
(57) Rutherford M, Srinivasan L, Dyet L, Ward P, Allsop J, Counsell S, et al. Magnetic resonance imaging in
perinatal brain injury: Clinical presentation, lesions and outcome. Pediatr Radiol 2006;36(7):582-592.
(58) Counsell SJ, Tranter SL, Rutherford MA. Magnetic Resonance Imaging of Brain Injury in the High-Risk
Term Infant. Semin Perinatol 2010;34(1):67-78.
(59) Shatrov JG, Birch SCM, Lam LT, Quinlivan JA, McIntyre S, Mendz GL. Chorioamnionitis and cerebral
palsy: A meta-analysis. Obstet Gynecol 2010;116(2 PART 1):387-392.
(60) Becroft DMO, Thompson JMD, Mitchell EA. Placental chorioamnionitis at term: Epidemiology and follow-
up in childhood. Pediatric and Developmental Pathology 2010;13(4):282-290.
(61) Schlapbach LJ, Aebischer M, Adams M, Natalucci G, Bonhoeffer J, Latzin P, et al. Impact of sepsis on
neurodevelopmental outcome in a swiss national cohort of extremely premature infants. Pediatrics
2011;128(2):e348-e357.
(62) Barkovich AJ, Miller SP, Bartha A, Newton N, Hamrick SEG, Mukherjee P, et al. MR imaging, MR
spectroscopy, and diffusion tensor imaging of sequential studies in neonates with encephalopathy. Am J
Neuroradiol 2006;27(3):533-547.
(63) Bonifacio SL, Glass HC, Vanderpluym J, Agrawal AT, Xu D, Barkovich AJ, et al. Perinatal events and
early magnetic resonance imaging in therapeutic hypothermia. Obstetrical and Gynecological Survey
2011;66(6):334-336.
(64) Glass HC, Glidden D, Jeremy RJ, Barkovich AJ, Ferriero DM, Miller SP. Clinical Neonatal Seizures are
Independently Associated with Outcome in Infants at Risk for Hypoxic-Ischemic Brain Injury. J Pediatr
2009;155(3):318-323.
(65) Miller SP, Newton N, Ferriero DM, Partridge JC, Glidden DV, Barnwell A, et al. Predictors of 30-month
outcome after perinatal depression: Role of proton MRS and socioeconomic factors. Pediatr Res 2002;52(1):71-
77.
(66) Benitz WE, Gould JB, Druzin ML. Risk factors for early-onset group b streptococcal sepsis: estimation of
odds ratios by critical literature review. Pediatrics 1999;103(6 I):1275.
(67) Jackson GL, Rawiki P, Sendelbach D, Manning MD, Engle WD. Hospital course and short-term outcomes
of term and late preterm neonates following exposure to prolonged rupture of membranes and/or
chorioamnionitis. Pediatr Infect Dis J 2012;31(1):89-90.
(68) Newton ER. Chorioamnionitis and intraamniotic infection. Clin Obstet Gynecol 1993;36(4):795-808.
(69) Modi N, Doré CJ, Saraswatula A, Richards M, Bamford KB, Coello R, et al. A case definition for national
and international neonatal bloodstream infection surveillance. Archives of Disease in Childhood: Fetal and
Neonatal Edition 2009;94(1):F8-F12.
(70) Newman TB, Puopolo KM, Wi S, Draper D, Escobar GJ. Interpreting complete blood counts soon after
birth in newborns at risk for sepsis. Pediatrics 2010;126(5):903-909.
(71) Inder TE, Wells SJ, Mogridge NB, Spencer C, Volpe JJ. Defining the nature of the cerebral abnormalities in
the premature infant: a qualitative magnetic resonance imaging study. J Pediatr 2003 8;143(2):171-179.
(72) Benitz WE. Adjunct laboratory tests in the diagnosis of early-onset neonatal sepsis. Clin Perinatol
2010;37(2):421-438.
(73) Dumoulin CL, Rohling KW, Piel JE, Rossi CJ, Giaquinto RO, Watkins RD, et al. Magnetic resonance
imaging compatible neonate incubator. Concepts in Magnetic Resonance Part B: Magnetic Resonance
Engineering 2002;15(2):117-128.
(74) Rutherford M, Ramenghi LA, Edwards AD, Brocklehurst P, Halliday H, Levene M, et al. Assessment of
brain tissue injury after moderate hypothermia in neonates with hypoxic-ischaemic encephalopathy: a nested
substudy of a randomised controlled trial. The Lancet Neurology 2010;9(1):39-45.
(75) Bayley N. Bayley Scales of Infant Development, Technical Manual. 2nd ed. San Antonio: The
Psychological Corporation; 1993.
(76) Bayley N. Bayley Scales of Infant and Toddler Development, technical manual. 3rd ed. San Antonio: The
Psychological Corporation; 2005.
25
(77) Anderson PJ, De Luca CR, Hutchinson E, Roberts G, Doyle LW, Callanan C, et al. Underestimation of
developmental delay by the new Bayley-III scale. Archives of Pediatrics and Adolescent Medicine
2010;164(4):352-356.
(78) Vohr BR, Stephens BE, Higgins RD, Bann CM, Hintz SR, Das A, et al. Are Outcomes of Extremely
Preterm Infants Improving? Impact of Bayley Assessment on Outcomes. J Pediatr (0).
(79) Lowe JR, Erickson SJ, Schrader R, Duncan AF. Comparison of the Bayley II mental developmental index
and the Bayley III cognitive scale: Are we measuring the same thing? Acta Paediatrica, International Journal of
Paediatrics 2012;101(2):e55-e58.
(80) Hajnal BL, Sahebkar-Moghaddam F, Barnwell AJ, Barkovich AJ, Ferriero DM. Early prediction of
neurologic outcome after perinatal depression. Pediatr Neurol 1999;21(5):788-793.
(81) Resnick MB, Gomatam SV, Carter RL, Ariet M, Roth J, Kilgore KL, et al. Educational disabilities of
neonatal intensive care graduates. Pediatrics 1998;102(2 I):308-314
26
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.