Int. J.

Devl Neuroscience 26 (2008) 311

www.elsevier.com/locate/ijdevneu

An adverse intrauterine environment: implications for injury

and altered development of the brain
Sandra Rees a,*, Richard Harding b, David Walker c
a

Department of Anatomy and Cell Biology, University of Melbourne, Victoria 3010, Australia
b
Department of Anatomy and Developmental Biology, Monash University 3800, Australia
c
Department of Physiology, Monash University 3800, Australia
Received 10 July 2007; received in revised form 30 August 2007; accepted 30 August 2007

Abstract
Abnormal development of the brain during fetal life is now thought to contribute to the aetiology of many functional and behavioural disorders
that manifest throughout life. Many factors are likely to underlie such abnormal development including genetic makeup and an adverse intrauterine
environment. This review will focus on prenatal hypoxicischemic injury and inflammatory/infective insults. A range of experimental models have
been used to characterise lesions formed in response to these insults and to determine mechanisms of damage resulting from such events. Relatively
brief periods of fetal hypoxia result in neuronal death (cerebellum, hippocampus, and cerebral cortex), white matter damage and reduced growth of
neural processes. These effects are more profound at mid than late gestation. Chronic mild placental insufficiency can result in fetal growth
restriction and deficits in neural connectivity and myelination. Exposure of the preterm fetus to inflammatory agents causes brain damage
particularly in the white matter and this is exacerbated by hypoxia. These studies show that the timing, severity and nature of specific insults are
critical in determining the pattern of injury and thus the extent to which neurological function will be affected postnatally. Defining the causes,
patterns and mechanisms of brain injury is crucial if we are to develop rational neuroprotective strategies to reduce the burden of altered brain
growth and poor functional and behavioural outcomes.
# 2007 ISDN. Published by Elsevier Ltd. All rights reserved.
Keywords: Perinatal hypoxia; Inflammation/infection; Brain injury; Therapeutic intervention

Protecting the developing brain during the fetal, peri-partum

and neonatal periods is presently one of the most significant
challenges in perinatal medicine. The prevention of brain injury
is made more urgent by the increasing number of preterm births
and the increasing survival rates of extremely premature
infants. Up to 10% of surviving infants will develop spastic
motor deficits such as cerebral palsy (Holling and Leviton,
1999) and a further 2550% will suffer developmental or
behavioural disabilities (e.g. Hack and Fanaroff, 1999; Volpe,
2001). Currently, there are no universally accepted treatment
regimens, perhaps due to our incomplete understanding of the
mechanisms underlying the etiology of such injury.

Abbreviations: BDNF, brain derived neurotrophic factor; ERG, electroretinogram; IL, interleukin; IUGR, intrauterine growth restriction; LPS, lipopolysaccharide; NMDA, N-methyl-D-aspartate; PPI, prepulse inhibition; ROS,
reactive oxygen species; TNF, tumour necrosis factor; VLBW, very low birth
weight.
* Corresponding author. Tel.: +61 3 8344 5797; fax: +61 3 9347 5219.
E-mail address: s.rees@unimelb.edu.au (S. Rees).
0736-5748/$34.00 # 2007 ISDN. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijdevneu.2007.08.020

Both human and animal studies have demonstrated that the

nature of the fetal neuropathology depends on the severity of
the insult and the gestational age of the fetus at the time of the
insult. In term infants, injury to neurons predominates after an
hypoxic insult; neurons of the CA1 region of the hippocampus,
cerebellar Purkinje cells and the deeper layers of the cerebral
cortex are the most frequently injured (Rivkin, 1997; Volpe,
2001). Cortical necrosis is prominent in the depths of the sulci,
possibly reflecting the relative avascularity of these regions (see
Volpe, 2001).
In contrast, in premature infants oligodendroglial/white
matter damage is the major injury. This distinctive lesion,
referred to as periventricular leukomalacia (PVL), includes
cystic infarcts adjacent to the lateral ventricles and gliosis
extending more diffusely throughout the cerebral white matter
(Banker and Larroche, 1962). PVL is often seen in cerebral
palsy victims especially after very preterm birth (Kuban and
Leviton, 1994). Recent magnetic resonance imaging studies
have demonstrated that diffuse white matter injury is very
common in premature infants while focal necrosis now occurs

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

rarely, in only about 5% of infants (Inder et al., 2003). It is

increasingly recognized that grey matter is also injured
including the cerebral cortex (Inder et al., 1999), hippocampus
(Isaacs et al., 2000) and cerebellum (Allin et al., 2001). It
remains unclear if this is a primary injury or secondary to the
injury in the underlying white matter. As damage to the cerebral
white matter will invariably interrupt afferent and efferent
cortical connections it is not unexpected that there will be
ensuing cortical neuronal damage either as a direct result of
axonal damage or subsequent to neuronal de-afferentation.
Furthermore, as pointed out by Limperopoulos et al. (2005) for
the relationship between prenatal growth of the cerebrum and
cerebellum, atrophy in one brain region can arise secondarily to
damage at another remote but connected region; this
diaschisis reflects the important trophic interactions that
exist between cells in the brain even though they might be
separated by a considerable distance.

cerebral vascular resistance, or in the case of maternal smoking,

an increase in the proportion of carboxyhemoglobin in the fetal
blood.
Although systemic fetal hypoxemia is regarded as a factor
usually associated with brain damage (McLennan et al., 1999;
Volpe, 2001) it is still not certain how this actually occurs in
human gestation and whether it is hypoxia alone or hypoxia in
combination with other physiological changes that render the
fetal brain vulnerable to damage. As we have indicated above,
placental insufficiency, perturbations to the fetal/placental
blood flow and perhaps also adverse fetal cardiac reactions to
the presence of proinflammatory or cytotoxic substances in the
fetal circulatation could compromise cerebral perfusion and
delivery of oxygen to the brain. The lingering uncertainty as to
how the catastrophic brain damage is actually brought about in
the human fetus makes it difficult to decide on the most
appropriate animal models of pregnancy-induced fetal brain
damage as we will discuss below.

1. Hypoxia and fetal brain injury

2. Maternalfetal infection and brain injury
During pregnancy the fetus is totally dependent on the
placenta for its supply of oxygen and nutrients. Interruption to
this supply can occur acutely by umbilical cord occlusion or
more chronically due to impaired placental function resulting
from maternal hypertension, maternal tobacco smoking, partial
placental detachment, placental villus edema or uterine artery
occlusion (Naeye et al., 1989). Systemic hypoxia is likely to be
a critical factor adversely affecting the fetal brain under such
intrauterine conditions although the delivery of oxygen and
perhaps other nutrients such as glucose and amino acids with
downstream effects on immature neurons and neuroglia cannot
be ruled out. Reductions in fetal cerebral oxygenation could
occur as a result of fetal hypoxemia, a reduction in fetal cerebral
perfusion due to a failure of cerebralhypoxic vasodilatation,
cardiac decompensation or in the worst case perinatal stroke.
The insult to the brain is commonly referred to as hypoxia
ischemia although it is still unclear how severely underperfused
the brain needs to be before irreversible brain damage occurs.
Experimental interventions, in which there has been no specific
interference with fetal cerebral perfusion such as maternal
hypoxia, fetal asphyxia, or fetal exposure to endotoxins, do not
result in dramatic decreases in fetal cerebral blood flow. Indeed
cerebral blood flow might even increase due to reflex
vasodilatation and to direct effects on the fetal cerebral
endothelium (Pearce, 2006). Furthermore, from mid-gestation
it is likely that autoregulatory mechanisms are sufficiently well
developed to allow for stabilisation of cerebral blood flow under
conditions where perfusion pressure varies (Szymonowicz
et al., 1990). Where reduction of fetal cerebral blood flow has
been observed, this has occurred in conjunction with decreased
cerebral metabolism (Blood et al., 2003) and therefore indicates
that cerebral metabolism and perfusion remain appropriately
coupled. Thus we posit that, in the fetus, the conditions that
ultimately lead to brain injury may not be ischemia per se, but
will include a reduction in the delivery of oxygen and/or
nutrients such as glucose and other events such as reductions in
placental blood flow, fetal arterial blood pressure, increased

Recent studies have indicated that maternal or intrauterine

infection/inflammation also play critical roles in perinatal brain
damage (Dammann and Leviton, 1997). Clinical and epidemiological studies (Dammann and Leviton, 1997) have shown
significant associations between maternal infection, neonatal
brain damage and increased levels of proinflammatory
cytokines in the amniotic fluid (Yoon et al., 1997), umbilical
cord plasma (Yoon et al., 1996) and brain (Deguchi et al., 1996).
The correlation between infection, white matter damage and
cerebral palsy is still being debated particularly for preterm
infants (Nelson et al., 2003).
Most commonly, invading microorganisms are thought to
gain access to the amniotic cavity and the fetus by ascending
from the vagina and cervix (Romero et al., 2003). Here they
induce the innate immune response and cause inflammation of
the chorioamniotic membranes and the production of proinflammatory cytokines. The cytokines and/or other inflammatory mediators then gain access to the fetus via swallowed
amniotic fluid or fetal lungs, eyes or nasal membranes. It has
been suggested that these agents increase the permeability of
the bloodbrain barrier with enhanced leukocyte infiltration of
the brain mediated by brain chemokines. Brain microglia and
astrocytes will be stimulated to upregulate cytokines and brain
injury will ensue. Furthermore, inflammation can interrupt
hemodynamic stability so that it is likely that hypoxic and
inflammatory pathways will interact to augment brain damage
as will be discussed later.
3. Possible role of placental infection in brain damage
There is some evidence from preterm births that chronic
placental infection may alter placental vascular structure but
may not impair fetal oxygenation (Salafia et al., 1995). In
contrast, acute infection may adversely affect placental
function leading to fetal hypoxemia and even hypoxic fetal
death (Garnier et al., 2001; Dalitz et al., 2003). Indeed the

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

presence of the placenta may be a key factor in the etiology of

perinatal brain damage, since the placenta itself has inflammatory reactions to bacterial or viral infection. The importance
of placental reaction to infection was recently shown by the fact
that small doses of LPS instilled into the utero-placental
circulation of pregnant sheep resulted in microglial activation,
macrophage infiltration and increased numbers of acidophilic
cells in the fetal brain, even though no endotoxin could be
detected in the fetal circulation (Hutton et al., in press). Thus
fetal brain damage might arise not only because oxygen
delivery to the brain is impaired but because of the
accumulation of reactive products in the fetal circulation
released from the placenta that affect fetal cerebral vascular
resistance and cerebral metabolism. Additional effects might
include changes of fetal blood volume, arterial pressure and
cardiac function. The fact that it is still necessary to speculate
on the role of the placenta and on fetal physiological parameters
in general, in promoting brain damage, is a reflection of our
ignorance as to precisely what is happening during pregnancy
to cause brain damage.
4. Animal models of intrauterine compromise
Advances in imaging techniques (Inder et al., 2003) are
helping to define neonatal injury in the developing human brain
but animal models are essential to identify the causative
mechanisms and to develop neuroprotective therapies. Several
recent publications (Northington, 2006; Back et al., 2006;
Derrick et al., 2007) have drawn attention to the fact that for
animal models to closely replicate possible scenarios of human
brain injury the insult needs to be delivered prenatally.
Epidemiological studies have shown that, at least in relation to
cerebral palsy, the etiology of 7080% of cases is likely to be
due to adverse antepartum and not intra- or post-partum events
(Nelson and Ellenberg, 1986). It is still contentious as to
whether overt brain injury is acquired before birth (Cowan
et al., 2003). Antenatal factors could initiate causal pathways
for brain injury that in association with a genetic predisposition
to hypoxicischemic damage might make some infants
particularly susceptible to the stress of parturition; brain injury
would continue evolving after birth. Many studies that have
focused on postnatal models of ischemia and/or global hypoxia
(e.g. Rice et al., 1981) do not take this cascade of events into
account. While these models have provided a valuable
paradigm for developing an understanding of mechanisms
involved in brain damage they do not allow for the influence of
the unique prenatal environment which, as we have pointed out
above, is likely to include important placental reactions to
hypoxia, hypoperfusion and inflammation. Furthermore, the
paucity of white matter in the immature neonatal rodent brain
precludes a close replication of the human lesion; neither does
the paradigm result in a clear motor phenotype. However
rodents do have the advantage of being amenable to postnatal
behavioural testing and the mouse in particular will allow
investigation of specific gene pathways.
Ideally, to model human brain damage one needs a species in
which the following criteria are met: a similar proportion of

brain development occurs in utero; an insult can be delivered in

utero at an equivalent stage of development identified to be
vulnerable in humans; the volume of white to grey matter is
similar to the human brain; the physiological outcome of the
insult can be monitored in utero and neurobehavioural
parameters can be tested after birth. Using these criteria all
species fall short, even non-human primates where the brain at
birth is significantly more developed than in humans. Species
which fulfil some of these criteria viz. sheep, rabbits and
guinea pigs will be discussed below. In choosing a species for
experimentation it is of course necessary to take into account
the timing of the insult with respect to the stage of brain
development achieved by the animal in question.
Over the past two decades we and others (e.g. Bocking et al.,
1988; Williams et al., 1992; Penning et al., 1994; Mallard et al.,
2003) have used ovine models which fulfil many of the criteria
described above with the exception that the brain is more
advanced at birth than in humans and behavioural testing
postnatally is challenging but not out of the question (Camm
et al., 2000). We point out that it has not necessarily been our
aim to produce a profound motor phenotype with the various
ovine models we have developed but rather functional or
behavioural deficits. The lack of any quantitative description of
neonatal behaviour in sheep to use as a reference against which
brain function can be assessed is currently a disadvantage.
Sheep however have a considerable advantage over smaller
species in that vascular catheters can be chronically implanted
to allow fetal blood sampling and to control alterations in the
intrauterine environment; furthermore they exhibit cerebral
hemodynamics similar to humans. Sheep have a long gestation
(term 147 days) making it possible to study responses to
insults over an extended period of brain development within the
intrauterine environment. Importantly, fetal sheep have a large
volume of white matter in which neuropathologies develop
which more closely resemble human neuropathologies than in
rodents.
We have used ovine models to examine the effects of a
reduction in the supply of oxygen and nutrients to the fetus, (a)
for 12 h, near mid-gestation (Rees et al., 1997, 1999), (b) brief
single (Duncan et al., 2004a) or repeated umbilical cord
occlusions in late gestation (Loeliger et al., 2003; CastilloMelendez et al., 2004), (c) chronic placental insufficiency
(2030 days) in late gestation (Mallard et al., 1998; Nicholls
et al., 2001a,b; Duncan et al., 2004b) and (d) maternal
carunclectomy which reduces the available sites for placentation, resulting in growth restricted fetuses that are chronically
hypoxemic, hypoglycaemic and have an altered endocrine
status (Rees et al., 1988).
We have also used a guinea pig model of chronic placental
insufficiency induced by uterine artery ligation during the
second half of gestation (term 67 days) (Nitsos and Rees,
1990; Mallard et al., 1999; Rehn et al., 2004). Guinea pigs also
have the advantage of a relatively long gestation allowing for
intrauterine manipulations at specific developmental stages and
they are amenable to postnatal behavioural and functional
testing. An advantage of their relatively advanced brain
development at birth, especially with respect to the develop-

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

ment of white matter tracts and myelination, is that it

compresses the time over which an intrauterine insult an be
assessed postnatally. Like rodents however guinea pigs have a
paucity of white matter.
Recently, a rabbit model of severe placental insufficiency
has been developed (Derrick et al., 2004); a level of intrauterine
monitoring can be achieved and the evolving brain damage can
be followed with sequential neuroimaging. The rabbit brain is
not as mature as the human brain at birth but this model has
been the first to result in a motor phenotype as a result of an
intrauterine insult and we will return to this finding later.
5. Which regions of the brain are damaged?
Our studies in both sheep and guinea pigs have focused on
the hippocampus, a region involved with learning and memory;
the cerebellum and striatum which are essential for the coordination and strategies involved in the execution of movement; the cerebral cortex (particularly the motor cortex) which
is involved in the initiation of movement; the cerebral white
matter which contains axon tracts connecting the cortex with
other parts of the brain; and the retina in the eye. The emphasis
on examining these brain regions in experimental animals
obviously arises from the observations in human infants who
display frank motor abnormalities (i.e. all the common forms of
spasticity) or who fail to mature sufficiently in childhood to
acquire language, learning and cognitive skills.
These studies have allowed us to reach the following
generalisations:
Acute insults in early gestation, resulting in a rapid drop in
oxygen delivery to the brain at a time when neurogenesis and
neural migration are at their peak, can result in the death of
neurons including Purkinje cells in the cerebellum, pyramidal
cells in the hippocampus and cortical neurons (Rees et al.,
1999) and a slowing of neural migration, at least in the
hippocampus (Rees et al., 1997). The growth of neural
processes is significantly retarded in the long term if the
neurons are particularly immature at the time of the insult (as in
the cerebellum) but will recover, after an initial delay, if axonal
and dendritic outgrowth is well established at the time of the
insult (as in the hippocampus). Acute insults can also cause
damage to the white matter resulting in diffuse injury and cystic
lesions in the periventricular area, neuropathologies resembling
those observed in cerebral palsy. Significantly, in our studies,
lesions occurred most prominently in fetuses from multiple
pregnancies, a risk factor for cerebral palsy. Fetal hypotension
was not a necessary accompaniment to the development of such
lesions (Rees et al., 1997).
Acute insults (umbilical cord occlusion) in late gestation
result in neuronal death in the cerebral cortex and striatum
(Loeliger et al., 2003), whereas hippocampal and cerebellar
neurons do not appear to be affected at the gross level. The
white matter is damaged but less extensively than when insults
are delivered earlier in gestation (albeit in our experimental
paradigm the earlier insult was more prolonged). These
observations support the finding that white matter injury is
more commonly seen in preterm than term infants; this appears

to be due to the vulnerability of immature oligodendrocytes to

hypoxemia (Back et al., 1998). Whether such damage persists
or becomes progressively worse by term is unknown in most
cases.
Chronic insults ( placental insufficiency) result in outcomes
which differ from acute insults in several aspects. Sheep fetuses
compromised throughout gestation (Rees et al., 1988), for 20
days in late gestation (Nicholls et al., 2001a,b; Duncan et al.,
2004b) or guinea pig fetuses compromised for the second half
of gestation (Nitsos and Rees, 1990; Mallard et al., 1999), are
growth restricted. The brain, although relatively spared in
relation to other organs, is reduced in weight. There is no overt
white matter damage although axonal myelination is reduced in
the central nervous system (Nitsos and Rees, 1990); this could
indicate a reduction in the number of myelinating glia and/or
the restricted capacity of those which form to generate myelin.
Thinner myelin sheaths affect axonal conduction velocity
which could contribute to altered neural function in growthrestricted individuals.
Following these chronic insults the growth of neural
processes and synaptogenesis is compromised globally in the
brain at term in the sheep (Rees et al., 1988; Rees and Harding,
1988) and guinea pig (Mallard et al., 1999; Dieni and Rees,
2005) and persists into young adulthood at least in some regions
of the brain (Rehn et al., 2004; Duncan et al., 2004b). Neurons
generally seem to survive chronic, mild intrauterine compromises however some populations are affected; reduced cell
numbers could relate to a direct effect of hypoxia on
neurogenesis or alternatively to cell death of postmitotic cells.
For example, in sheep after 20 days of chronic placental
insufficiency in late gestation, dopaminergic amacrine cells
(interneurons) in the retina, which are involved in contrast
sensitivity, are reduced in number but other classes of amacrine
cells are not affected (Duncan et al., 2004b). The loss of even
small numbers of specific classes of cells could significantly
affect particular neural functions. In the studies where we have
been able to examine the structure of the brain and retina
postnatally in the long term (Duncan et al., 2004b), we have
found that some alterations persist (reduction in dopaminergic
amacrine cells) others resolve (dendritic and axonal growth in
the cerebellum catches up) and still others develop after birth
(reduction in dendritic growth of CA1 hippocampal neurons)
demonstrating the importance of long-term follow-up studies in
developing a complete understanding of the effects of a prenatal
insult.
In the guinea pig model of chronic placental insufficiency,
we have observed enlargement of the lateral ventricles most
likely resulting from reduced growth of neural processes and
reduced neuronal numbers in some brain regions (Mallard et al.,
1999). Ventriculomegaly is one of the most consistent findings
in the brains of patients with schizophrenia (Johnstone et al.,
1976; Hopkins and Lewis, 2000). Our study demonstrated
experimentally for the first time that such alterations can
originate from an insult in utero (Mallard et al., 1999) and
persist into adolescence (Rehn et al., 2004). Furthermore, we
have also demonstrated in this model, reduced brain weight,
reduced basal ganglia volume, the absence of astrogliosis, a

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

significant delay in the development of the oligodendrocyte

lineage, a reduction in white matter volume and sensorimotor
gating deficits (discussed below) at adolescence; these deficits
parallel to an extent, the situation in some patients with
schizophrenia (Rehn et al., 2004). Although no animal model of
a complex human disorder is ever likely to emulate deficits in
all aspects of structure and function observed in patients with a
neuropsychiatric illness, our findings support the early
neurodevelopmental hypothesis of schizophrenia (Akil and
Weinberger, 2000). A strength of this model is that it mimics a
condition which could occur in human pregnancy, albeit at the
more severe end of the spectrum of prenatal conditions. Other
models in neonatal animals such as N-methyl-D-aspartate
receptor (NMDA) blockade (Harris et al., 2003), lesioning of
hippocampal pathways (Lipska et al., 1995) and depletion of
dopamine, support the neurodevelopmental hypothesis but do
not replicate specific perinatal events.
6. How might prenatal brain damage occur in
compromised fetuses?
The etiology of the brain damage described above is likely to
be multifactorial; however as we have indicated, reduced
oxygen delivery and its downstream effects are likely to play a
major role. The principal pathways leading to cell death after
hypoxia and ischemia are initiated by energy depletion
followed by some or all of following events: increased
neuronal release and reduced uptake of glutamate by glia;
activation of glutamate receptors; accumulation of cytosolic
calcium; activation of a variety of calcium-mediated deleterious events including generation of reactive oxygen species
(ROS) such as superoxide anion, hydroxyl radicals and nitric
oxide derivatives. ROS interact with the lipid components of
cellular membranes, initiating lipid peroxidation which results
in the breakdown of lipid constituents into highly reactive byproducts including lipid aldehydes, for example hydroxynonenal and malondialdehyde. These reactive aldehydes then bind
to and modify protein creating protein adducts. Nitrosative
stress results from nitric oxide (NO) released from reactive
microglia reacting with superoxide anions to form peroxynitrite
which targets tyrosine residues of proteins to form nitrotyrosine
residues. Both of these processes are highly damaging to cell
membranes. These events can result in mitochondrial disruption and immediate or delayed cell death, with the cascade of
damaging events unfolding over hours to days after the primary
insult (see Johnston et al., 2000).
The late precursor cells (pre-oligodendrocytes) and immature oligodendrocytes that predominate in the developing
periventricular white matter during the period of highest risk for
white matter damage in humans (2332 weeks of postconceptional age) are particularly susceptible to hypoxiaischemia.
Studies have shown that markers of oxidative and nitrosative
stress can be detected in areas of periventricular white matter
damage (Haynes et al., 2003). The sensitivity of these cells to
injury is associated with a limited capacity to resist oxidative
stress (Back et al., 1998) and the expression of Ca2+ permeable
AMPA receptors on their somata (Fern and Moller, 2000) and

NMDA receptors on the processes of immature oligodendrocytes (Salter and Fern, 2005). Recent studies suggest that
glutamate released during hypoxiaischemia from late precursors and immature oligodendrocytes and axons can activate
glutamate receptors on adjacent oligodendrocytes leading to
injury (Back et al., 2007). Riddle et al. (2006) have
demonstrated in the immature fetal sheep that during severe
cerebral hypoperfusion white matter damage is not uniform but
rather coincides with the presence of susceptible populations of
oligodendrocyte progenitors. In regions of diffuse white matter
injury caused by LPS exposure we have observed a reduction in
oligodendrocyte numbers, damage and attenuation of the
processes of the surviving oligodendrocytes and destruction of
axons (Duncan et al., 2002; Nitsos et al., 2006).
The mechanisms underlying axonal damage are not certain
but could involve glutamate receptor activation and a toxic
influx of Ca2+. In the ovine fetus we have shown that the level of
glutamate efflux in white matter positively correlates with the
degree of damage to the white matter (Loeliger et al., 2003).
Axonal damage could also occur as a result of reverse operation
of ionic pumps. Energy depletion causes failure of Na+K+
ATPase which allows an unopposed inward leakage of Na+ and
membrane depolarization acts to drive the Na+Ca2+ exchanger
in the Ca2+ import mode, leading to intracellular Ca2+ overload.
Excess Ca2+ is expected to disrupt mitochondrial function,
leading to axonal damage (Stys et al., 1991). If the insult is less
severe, a sub-lethal influx of Ca2+ could significantly retard the
growth of axons.
The role played by hypoxia during a primarily inflammatory
insult is still uncertain. We know that when the LPS is
administered to the fetus either via a prolonged intravenous
infusion (Duncan et al., 2006) or intra-amniotically (Nitsos
et al., 2006), fetal hypoxemia does not ensue and the resultant
brain damage is not as significant as when LPS is administered
acutely, in repeated boluses, a treatment regimen which results
in fetal hypoxemia, hypotension and hence reduced cerebral
oxygen delivery (Duncan et al., 2002). From previous studies
we know that predominantly hypoxic insults (without fetal
hypotension) (Rees et al., 1997) also cause white matter
damage but that it is not as reproducible or generally as severe
as that resulting from LPS exposure. Thus it appears that there
are synergistic pathways between hypoxia and inflammation
which potentiate the evolution of brain damage. In accordance
with this notion LPS has been shown to sensitize the immature
rat brain to hypoxicischaemic injury (Eklind et al., 2001).
Whether this augmentation of damage applies to human
pregnancies is not yet certain but it is known that when
intrauterine inflammation is accompanied by fetal asphyxia
there is a dramatic increase in cerebral palsy (Nelson and
Grether, 1998).
Clearly, the etiology of the fetal brain damage in
inflammation is multifactorial and is likely to include an
increase in circulating cytokine levels. We have shown, for
example, that TNF-a (Dalitz et al., 2003) and IL-6 levels
(Duncan et al., 2002) increase within 6 h of LPS-exposure. It
has been proposed that circulating cytokines might act on
cerebral endothelial cells or perivascular cells to upregulate

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

prostaglandin synthesis resulting in increased bloodbrain

barrier permeability (Yan et al., 2004); thus the administration
of the endotoxin lipopolysaccharide (LPS) to fetal sheep results
in the extravasation of plasma proteins and macrophages into
the brain (Yan et al., 2004). The powerful oxidative activity of
macrophages, also a characteristic of the metabolism of
activated microglia, not only adds to the burden of increased
ROS and proinflammatory cytokines in the brain, but it is likely
to result in the accumulation of quinolinic acid (Heyes et al.,
1996), a glutamate receptor agonist that with prolonged
exposure has been shown to be neurotoxic (Stone, 1993).
Quinolinic acid, a metabolite of tryptophan is known to be
released from the human placenta (Manuelpillai et al., 2003)
and is present in higher concentrations in cord blood of babies
from pregnancies with clinical signs of infection (Manuelpillai
et al., 2004). It is present in fetal sheep blood after LPS
treatments (Yan et al., 2004), and is increased in blood and brain
of chronically growth-retarded fetuses (Nicholls et al.,
2001a,b). The activation of microglia by low grade infection
in pregnancy and release of quinolinic acid amongst other
molecules may be one of the ways in which the fetal brain is
made more vulnerable to the effects of hypoxia, whether this
occurs subsequently in utero, during labor or birth, or in the
postnatal period when respiratory function may be sub-optimal.
Fetal nutrient restriction and/or altered endocrine and
growth factor status may also play a role in brain damage,
particularly when the insult is more prolonged. For example, we
have shown that the neurotrophin brain derived neurotrophic
factor (BDNF) is significantly reduced in chronic placental
insufficiency in the fetal hippocampus (Dieni and Rees, 2005)
and retina (M. Loeliger pers. comm.). Thyroid hormone and
insulin-like growth factor both known to play an essential role
in neuronal and neuroglial development, are also reduced in
chronic placental insufficiency (see Nitsos and Rees, 1990).
7. Altered brain function in prenatally compromised
offspring
Evidence of changes in the structure of the brain are
informative but they need to be supported by studies of brain
function in compromised individuals in order to obtain a clearer
picture of how altering the normal course of brain development
can affect function, particularly in the long term. As indicated
above, it is not the goal of all models of prenatal compromise to
produce a severe motor phenotype. In our guinea pig model of
chronic placental insufficiency which results in growth
restriction, we have been interested in testing more subtle
neurobehavioral and functional outcomes. This is relevant to
the observation that babies of low or very low birth weight
(VLBW), many of whom are also growth restricted, are at risk
for long term auditory and visual impairment including
sensorineural hearing loss and deficits in visual acuity, colour
vision and contrast sensitivity (Dowdeswell et al., 1995).
In the guinea pig we have shown that there are long-term
alterations in retinal function as detected by measuring the
electroretinogram (ERG) in young adults (Bui et al., 2002).
We have detected changes in the ERG in IUGR animals,

indicative of subtle changes to photoreceptors and amacrine

cells. Such effects could contribute to the visual impairment
reported in VLBW infants. In the same animal model we have
tested the auditory system by giving an acoustic stimulus and
recording the auditory brainstem response. We have
identified subtle differences in neural conduction in auditory
pathways in the prenatally compromised young adult animals
compared to controls (Rehn et al., 2002). This might reflect
alterations in the degree of myelination of auditory brainstem
pathways or changes in synaptic efficacy. These changes
might underlie hearing loss and speech perception abilities in
VLBW and/or growth restricted children (Anagnostakis et al.,
1982).
Furthermore, in the prenatally compromised guinea pig we
have demonstrated a reduction in prepulse inhibition (PPI) of
the startle response (Rehn et al., 2004), a behavioural paradigm
which is thought to indicate the ability of the central nervous
system to filter out extraneous stimuli; PPI is reduced in
patients with schizophrenia and in some other psychiatric
disorders. This result indicates that adverse prenatal conditions
lead to long-term alterations in function which resemble
alterations in patients with schizophrenia and hence adds
further support to the early neurodevelopmental hypothesis of
schizophrenia.
Recently in the rabbit model of acute utero-placental
ischemia near term mentioned above, abnormalities in motor
control and hypertonia were demonstrated in neonatal offspring
(Derrick et al., 2004). In some ways this is a landmark study, the
first model of antenatal uterine hypoxiaischemia which results
in motor deficits and neuropathology with some parallels to
the deficits seen in cerebral palsy. The study revealed that the
duration of the hypoxiaischemia was critical with durations
less than 37 min having little effect but those more than 37 min
having substantial effects. It is possible that some subtle deficits
were produced by hypoxicischemic insults <37 min that will
not be detected if the presence of gross motor abnormalities is
considered as the only significant end-point of such experiments.
8. Possible therapeutic intervention to prevent or
ameliorate injury and altered brain development
In order to be able to provide timely therapeutic intervention
for the fetus or infant at risk of brain damage we need to be able
to detect when an insult has occurred. This can be challenging
as it is possible for placental dysfunction and fetal brain injury
to occur without significant clinical indications being evident
during gestation (Grafe, 1994). Advanced neuroimaging is now
providing the opportunity to identify and then monitor the
evolution of brain injury in compromised fetuses and neonates.
Another approach is to identify reliable markers of brain
damage in the blood of newborns (Leviton and Dammann,
2002). The presence of proteins such as S-100B (from
astrocytes) or neuron-specific enolase (from neurons) is
considered as evidence that these cells have been damaged
and the proteins released into the blood through increased
permeability of the bloodbrain barrier.

S. Rees et al. / Int. J. Devl Neuroscience 26 (2008) 311

From clinical observations and animal experimentation

(Johnston et al., 2000) we now know that the cascade of
damaging events to the brain can unfold over several days; this
suggests that the window of opportunity for intervention is
longer than previously envisaged. In animal models attempts
have been made to inhibit the hypoxicischemic cascade at
almost every level with, for example: NMDA receptor
blockade; the use of nitric oxide synthetase inhibitors; blockade
of apoptosis by inhibition of caspases; blocking the upregulation of free radical formation. Other approaches include the
application of growth factors in utero (Johnston et al., 1996) the
prevention of energy depletion via mild hypothermia after birth
(Williams et al., 1997) and treatment of the maternal animal
with melatonin to prevent increased ROS formation (Miller
et al., 2005). Many of these approaches have been effective to
varying degrees in animals, but with the exception of
hypothermia in some cases (Gluckman et al., 2005), they
have been less successful or have had untoward side effects in
humans. Several agents are currently being tested as either
stand-alone therapies or in combination with hypothermia. For
example N-acetyl-cysteine, a free radical scavenging agent, has
proven effective in a postnatal model of endotoxin-sensitised
hypoxicischemic brain injury in rodents (Wang et al., 2007)
but causes adverse physiological reactions when administered
to LPS-exposed ovine fetuses (Rees et al., 2006). Erythropoetin, an anti-apoptotic and tissue protective cytokine, has also
been shown to be effective in rodent models of brain damage
(Ghezi and Brines, 2004) but its effectiveness in large animal
models is still being determined (Rees et al., 2007). Testing
therapeutic agents in appropriate animal models is obviously
crucial for the translation of therapies to the human infant.
There might be an opportunity to improve the neuronal
environment immediately after birth in neonates at risk to
enhance the growth of axons, dendrites and synaptogenesis and
myelination of axons. We know that these events can be
manipulated in the period of neonatal plasticity in the months
after birth. As described above, BDNF levels are decreased in
chronic placental insufficiency and the growth of processes is
reduced. Increasing the levels of growth factors or other
molecules immediately after birth, ideally by systemic injection
of small molecule mimetics of neurotrophins or growth factors
which can cross the blood brain barrier, might be successful in
restoring appropriate brain development and function. Such
manipulation could also produce aberrant or inappropriate
synaptogenesis so the outcome of such treatments, indeed of
any therapeutic treatment, needs to be tested for functional
improvement.
9. Concluding remarks
Growing evidence supports the notion that an adverse
intrauterine environment can lead to many of the neurological
and behavioural deficits which manifest after birth. The timing
and severity of prenatal insults are critical in determining the
nature of the outcome in terms of the severity of the damage and
the regions of the brain affected. In animal models it has been
demonstrated that relatively brief (acute) periods of fetal

hypoxemia can have significant effects on the fetal brain, for

example death of susceptible neuronal populations (cerebellum, hippocampus, cortex) and cerebral white matter damage.
These effects appear to be more profound in mid than late
gestation. Chronic mild placental insufficiency which includes
fetal hypoxemia, nutrient restriction and altered endocrine
status, can result in long term deficits in neural connectivity as
well as affects on postnatal function, for example in the
auditory and visual systems. Perinatal infection and cytokine
exposure is now considered to be an important factor in causing
brain damage; there are likely to be synergistic pathways
between hypoxia and inflammation which potentiate the
evolution of injury. A central factor in the many forms of
fetal brain injury appears to be a severe reduction in the delivery
of oxygen (and perhaps glucose) to the developing brain. A
greater understanding of the etiology of perinatal brain damage
will allow us to devise strategies to intervene and reduce the
burden of perinatal brain injury which is likely to increase with
the increasing survival rates of preterm infants. Creating the
most ideal extrauterine environment for preterm infants will
also be essential if we are to facilitate appropriate neural
development postnatally.
Acknowledgement
We acknowledge the support of the National Health &
Medical Research Council of Australia.
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