Semin Neonato11998;3:87-101

Pharmacological strategies for the prevention of

perinatal brain damage
Alistair J. Gunn and Peter D. Gluckman

School of Medicine, The University of
Auckland, Private Bag 92019, Auckland,
New Zealand
Key words: Hypoxic-ischaemic
encephalopathy, perinatal asphyxia,
neuroprotection, neuronal rescue,
neurotrophic factors
Recent clinical studies have confirmed that severe perinataI asphyxial injury is
associated with delayed development of cerebral energy failure from 6 to 15 h after
birth. Reversible hypoxic-ischaemic neural injury precipitates a cascade of injurious
biochemical events, which lead to delayed neuronal death. These damaging mechanisms
however are balanced by endogenous protective mechanisms that may help to limit the
final extent of injury. The delayed evolution of neural damage represents a window of
opportunity for possible treatment. The present review discusses possible pharmaco-
logical interventions, which may act either by directly blocking injurious factors or by
enhancing protective mechanisms. Extensive experimental data show the potential of
these agents to treat perinatal asphyxia, stroke, and other forms of acute brain injury.

Introduction mechanisms are hypoxic depolarization and accu-

Perinatal hypoxic ischaemic encephalopathy (HIE)
is the clinical manifestation of perinatal asphyxia.
Despite increasingly sophisticated prenatal and
postnatal care there has been no corresponding
change in the incidence of moderate to severe HIE,
typically I to 311000 live births, in recent years [1].
At the same time, fundamental advances in under-
standing the pathophysiology and biochemical and
cellular events have raised the possibility that it
may be possible to manipulate the biochemical
cascade leading to eventual cell death, and thus
improve outcome. The present chapter will outline
a number of novel experimental therapeutic inter-
ventions. Other chapters will focus on particular
factors, particularly the involvement of oxygen free
radical overproduction.
HIE is related to global (systemic) asphyxia;
there is strong evidence that actual cerebral injury is
precipitated by reduced cerebrovascular perfusion
secondary to cardiac compromise and systemic hy-
potension [2-4]. The cerebral insult is thus typically
global and reversible; thromboembolism in which
neuronal death has a different pathophysiological
basis is seldom implicated. During such global
hypoxic-ischaemic insults the key injurious cellular
1084-2756/98/020087+15 $12.00/0
mulation of excitotoxins, both of which promote
immediate cell swelling (cytotoxic oedema) and
excessive intracellular calcium entry [5-7]. During
reoxygenation after asphyxia further injury may be
linked to excessive oxygen free radical production.
Although cell death can occur during asphyxia
(primary cell death), these events, particularly intra-
cellular calcium exposure, may trigger secondary
pathologic processes leading to delayed to second-
ary neuronal death. The mechanisms involved in
the secondary phase include active cell death
(analogous to developmental apoptosis) [8, 9],
further exposure to excitotoxins [10, 11], and cyto-
toxic actions of activated microglia [12, 13]. The
associated seizures, cerebrovascular reactions and
cytotoxic oedema may also contribute to ongoing
injury [14, 15].
Protective endogenous cerebral responses may
to some extent help balance the injurious factors. In
addition to acute centralization of cardiac output
[16], several potential endogenous protective sys-
tems exist, including: neuromodulators/inhibitory
neurotransmitters [17], cellular and neurotrophic
factors [18], and cerebral cooling. There is some
evidence for example that part of the greater
resistance to hypoxia of the fetus and newborn
© 1998 W.B. Saunders Company Ltd
88 A.d. Gunn & P. D. Gluckman

may be related to much greater accumulation of

Primary Secondary Resolution
inhibitory neurotransmitters during asphyxia [11], )hase phase
and of neurotrophic factors [19] after injury. Delayed, post-ischaemic
Oc Latent seizures

The timing of cell death

Although some neurons and glia die during the
-r.r.l
primary phase, during or shortly after the asphyxial
insult, the seminal observation in the last decade is -15
that many cells at least partially recover but go on
to die many hours later. The longer and more -20 I , I , , ,, I I I I I
severe the insult, the greater the proportion of
primary neural injury [10, 21]. There is now good 160
experimental evidence for the occurrence of
150
delayed neuronal death both in immature and adult Secondary eytotoxie oedema
animals [21-24] and after cardiac arrest in man [25]. ~
¢.,
140
Consistent with these histological data, a ~130
secondary phase of deterioration has been clearly
•~ 120
demonstrated clinically in birth asphyxia. Some
asphyxiated infants have normal cerebral energy ~ 11o
o
metabolism shortly after birth as measured by rj
100
magnetic resonance spectroscopy (MRS), but then
90 I ~ I , , ,, I I I I I
show secondary failure of energy metabolism many
hours after birth, which reaches a maximum by Reperfusion ~ Secondary
48 h [26]. Subsequently, neuro-developmental out- .3~ 120 / hyperperfumon
come correlates with the degree of energy failure
[27]. Further supportive evidence for a significant ~ ,,I I
phase of secondary injury in man comes from 100' 1
measurements of cytochrome oxidase [28], the
development of cytotoxic oedema in neonatal
encephalopathy [29], and from electroencephalo- 80
graphic studies which confirm the delayed C9 /! IDelayed hypoperfusion
evolution of seizure activity after asphyxia [30]. 0
/,
I', I , I ~ , ,, I I I I I
Experimentally, a similar pattern has been shown 2h 6h 12h 24h 48h 72h 96h 120h

in studies of severe hypoxia-ischaemia in the piglet Carotid Time

[31], where the magnitude of delayed neuronal loss
was proportional to that of secondary energy
failure [24]. The evolution of cerebral injury after
severe ischaemia has been studied in an in utero
model in the late gestation fetal sheep, which is
independent of the confounding variables of acute
surgical stress and anaesthesia [32]. As illustrated in
Figure 1, there is often a latent period of general-.
ized neural depression and delayed hypoperfusion
for many hours after the insult (latent phase). The
secondary deterioration typically starts 6--24h
post insult and continues over several days [27, 31].
It is marked by post asphyxial neuroexcitation
which may manifest as seizures, cytotoxic oedema
[14] and enhanced cerebral blood flow [15, 33]. The
magnitude of the injury determines the magnitude
of the secondary phase and the degree of neuronal
occlusion
Figure 1. The time sequence of changes in mean cortical
impedance (a measure of cytotoxic oedema), cortical EEG
intensity and carotid blood flow after 30 min of global
cerebral ischaemia in fetal sheep 01=7), The insult (primary
phase) is marked by the dashed lines. Although the EEG
remains suppressed for many hours after this (latent phase),
with secondary hypoperfusion, cortical impedance rapidly
resolves within 30 min, with only a small residual
elevation. The secondary phase is shown by an abrupt
increase in EEG activity nearly 24 h after insult
(corresponding with intense epileptiform activity), with an
associated rise in cytotoxic oedema and carotid blood flow.
Data derived from Gunnet a]. (I997) [I5].
loss [20]. Using near infra-red spectroscopy (NIRS),
the major phase of loss of cytochrome oxidase (a
measure of mitochondrial failure) has been shown
to occur during the secondary period [33].
Prevention of perinatal brain damage
T a b l e 1. M e c h a n i s m s o f n e u r o n a l cell loss
Primary phase
Calcium cytoaccumulation
M e m b r a n e instability
O x y g e n flee radicals
Excitotoxicity
Apoptosis
Microglial r e s p o n s e
Types of cell death
Two morphological patterns of cell death are
seen--necrosis and apoptosis [21, 34, 35]. Typi-
cally, a high proportion of neuronal loss during the
secondary phase after perinatal hypoxia-ischaemia
is by apoptosis [36], as defined by shrinkage of
the cell, 'karyohexis', associated with specific
endonuclease-mediated DNA degradation [24]. The
relative proportion of necrotic cell death, as defined
by loss of plasma membrane integrity with a
random pattern of DNA degradation, is seen
relatively earlier with increasing severity of the
primary insult [21]. There is some limited evidence
that particular cell populations may be more prone
to apoptosis rather than necrosis after hypoxic-
ischaemic injury, particularly cerebral white matter
and less mature neuronal populations [37].
Apoptosis is a process of active cell death, and is
classically seen in the normal loss of excess cells
(including neurons) that occurs during develop-
ment [35, 38]. It may be initiated by several
intracellular pathways but the final events involve
alterations in the ratio of various intracellular
factors such as BCL, which inhibits apoptosis
[39], and Bax which promotes apoptosis [39, 40]
and to activation of proteases such as ICE
(interleukin-converting enzyme) [41, 42]. The final
events include intranuclear fragmentation and
endonuclease-mediated DNA fragmentation [21].
The distinction between apoptosis and necrosis
may not be clearcut. In developing rats subject to
unilateral hypoxic-ischaemic (HI) injury (using the
modified 'Levine' approach of unilateral carotid
ligation followed by inhalational hypoxia), a
delayed phase of neuronal death has been demon-
strated histologically [43]. Mild injury leads to
selective neuronal loss developing from 24 h after
the end of the insult [21]. Interestingly, in that
paradigm although severe hypoxia led to cell death
89
Reperfusion phase Latent phase Secondary phase
+ + + + ? 4-
+ + + + + + + - +
+ + + + - i
+ + + ? - + + +
-- -- + + + + ?+ +
- - - + +
with predominantly necrotic morphology, this
still showed a delayed evolution, starting from
approximately 10 h after hypoxia.
Types of treatment
Based on these concepts there are clearly two
general approaches to treatment: 'neuroprophy-
laxis' or treatment prior to or during the primary
phase; and 'neuronal rescue' where the strategy is
to inhibit or prevent the cascade of events leading
to secondary injury. The pathogenic factors likely
to be involved in each of the phases are shown in
Table 1. The latent period between the primary and
secondary phases creates a window of therapeutic
opportunity for rescue therapy. Neuroprophylaxis
is likely to be limited to selected situations such as
extracorporeal membrane oxygenation or coronary
bypass surgery. However it may have a role in
high risk obstetrics. During resuscitation, there
will still be a place for addressing the primary
mechanisms particularly OFR induced injury.
Potential pharmacological therapies include
blockade of excitotoxins and calcium entry,
antagonism of cytotoxins such as OFRs, or finally,
using endogenous neuroprotective factors to
suppress secondary events, such as inhibi-
tory neurotransmitters/neuromodulators and anti-
apoptotic therapy such as growth factors. Figure 2
illustrates the effect of selected putative prophy-
lactic or rescue agents on regional neuronal loss
after cerebral ischaemia.
Confounding factors
The effectiveness of any particular strategy will
depend not only on the injurious mechanisms
90
Prophylactic vs rescue therapy
100 /
80t-I7 *
60HII**
7ol-I T i between drug induced vasodilatation and asphyxial
%~ 40 I sion and secondary cerebral compromise [44].
oFI II
2o b l l •
oHi I
0 /I I I
GM-1 Flunarizine MK-801IGF-1 I-NNA
8O
70
I 1'* lili
m~ 50
m,-t
-~4 4o
%r~ 30
~ 20
2; 10
0 protective [49, 50], and that this can be produced,
GM-1 Flunarizine MK-801 IGF-1 1-NNA
Figure 2. Summary of the effects of selective prophylactic
(GM-I and Flunarizine, left) or rescue therapies (MK-80I,
IGF-1 and I-NNA, right) on neuronal damage in the
parasagittal cortex (top panel) or the CA1 subfield of the
hippocampus (lower panel), assessed 3 days after 30 rain
cerebral ischaemia in fetal sheep. In general the effect of
neuroprophylaxis was greater than that of rescue therapy.
GM-1 ganglioside [69], and Flunarizine [44], a calcium
channel antagonist, significantly reduced neuronal loss when
given prior to ischaemia. Interestingly, a higher dose of
Flunarizine was not protective, probably because of an
impaired fetal cardiovascular response. Post insult therapy
was not effective (unpublished data), Other agents have
been shown to provide at least partial neuroprotection post
insult. For example, a single dose of I u g rhlGF-l,
administered i.c.v. 2 h after, ischaemia had a modest effect
on damage in the parasagittal cortex with a greater
improvement in the hippocampus and lateral cortex [I50].
Although MK-80I, a potent highly selective NMDA
antagonist completely abolished post-ischaemic seizures, it
did not affect parasagittal cortical injury [88]. L-NNA, a
competitive antagonist of NOS attenuated the delayed
luxury perfusion, but tended to worsen the histological
outcome [105]. This adverse effect is likely to be mediated
by inhibition of endothelial NOS and consequent restriction
of cerebral blood flow. *P<0.05. **P<0.01. ([--1), control
ischaemia; ( • ) , treated.
operative in the brain at the time chosen, but also
on any other effects of the treatment. Many
experimental studies have used the approach of
combined hypoxia-ischaemia in the immature rat
to test proposed interventions, since it is much
more reproducible than systemic asphyxia. Studies
using this type of 'functional' experimental
approach have made critical contributions to our
A . J . Gunn & P. D. Gluckman
knowledge, however it is important to appreciate
their key limitation, which is that of necessity they
do not fully address possible adverse effects of
proposed therapies. One example might be
cardiac injury, which may cause further hypoten-
One other major issue to consider when evalu-
ating studies is temperature. It is well known that
small changes in temperature, of only a few
degrees, during and immediately after hypoxia-
ischaemia, can critically modulate damage [45].
This confounded early studies of antiexcitotoxic
therapy; the apparent benefits of NMDA antagon-
ists seem to have been either partly due to associ-
ated hypothermia [46], or even synergistically
increased by cooling [47, 48]. More recently it has
become clear that prolonged hypothermia of only
I to 2°C during the secondary phase may be
for example, by anti-excitotoxic agents [51].
The reciprocal issue arises with mild hyper-
thermia during the secondary phase, which occurs
in several species after stroke; unless this is con-
trolled, it may exacerbate damage and so mask real
treatment effects [52]. It is interesting to consider
whether the inevitable (except in utero) reduction
in brain temperature as a result of reduced cerebral
metabolic activity and blood flow during hypoxia-
ischaemia [53] may in itself be one endogenous
protective factor. The mechanism of action of post
insult cooling is unclear, but evidence has been
presented to suggest that it acts primarily to block
apoptosis [36]. If this is the case, it is likely that
combination therapy of hypothermia with agents
acting on other pathways will prove to be an
important modality in the future.
Prevention of reperfusion injury
Oxidative metabolism in the mitochondrial elec-
tron chain, as well as other oxidation-reduction
reactions in the cytoplasm normally produce oxy-
gen free radicals (OFRs), i.e. an oxygen molecule
that contains an uneven number of electrons in
its outer orbit ('02-). These species are highly
reactive, and if not neutralized can lead to degra-
dation of cell membrane lipids (by peroxidation
of the unsaturated fatty acids in the lipid bilayer)
[54] with intracellular swelling [55] and impaired
metabolism [56]. In addition, a reciprocal relation-
ship between oxygen free radical production and
Prevention of perinatal brain damage
calcium or excitatory amino-acid production has
been proposed. Oxygen free radicals increase
glutamate release [57], while the entry of extra-
cellular calcium has been shown to uncouple mito-
chondrial electron transport, and so to promote
free radical production [58].
Increased production of oxygen free radicals has
been implicated during asphyxia, and more particu-
larly during reoxygenation when they may exceed
scavenger enzyme activity [56, 59, 60]. One source
is accumulation of substrates of the xanthine
oxidase pathway as a result of utilization of ATP
during asphyxia. Hypoxanthine is oxidized to
xanthine, then to uric acid by the oxidase form of
xanthine oxidoreductase. Oxygen levels rise dur-
ing reperfusion, leading to a burst of reactive
oxygen metabolites. Another source of OFRs is
accumulation of neutrophils during reperfusion,
which produce lytic bursts of H202, nitric oxide
(NO) and other types of OFRs.
A number of free radical scavengers have been
identified. Allopurinol inhibits xanthine oxidase
and thus will reduce OFR production. A limited
protective effect has been found experimentally in
the 7-day-old rat 'Levine' preparation [61, 62].
Similarly, post hypoxic administration of deferox-
amine, which binds free iron, which catalyses the
production of OFRs, reduced cortical injury in the
same preparation [63]. Interestingly, in the adult
dog however, combination therapy with OFR
scavengers and anti-excitotoxic agents was
required to improve recovery after ischaemia [64].
The use of superoxidase dismutase as a therapeutic
agent has been suggested but appears to have a
narrow therapeutic range [56, 65]. Mannitol has
been claimed as an OFR scavenger although with
little evidence [66]. The other claimed role of
mannitol has been for the treatment of cerebral
oedema. However as the 'oedema' of brain injury is
intracellular or cytotoxic, a primarily osmotic
therapy would not be anticipated to be of great
value [67].
Agents such as GM1 ganglioside probably have
their major beneficial effects by incorporation into
the plasma membrane and thus increasing stability]
resistance to lipid peroxidation [68]. GM1 ganglio-
side is neuroprotective when given prior to or
during hypoxic-ischaemic injury in the fetal sheep
[69, 70] but not when given more than 5 min post
insult in the adult rat [71]. Given their apparently
excellent safety profile, and lipid solubility, this
class of reagents may well have a role in neuro-
prophylaxis [69, 70].
91
Calcium channel antagonists
There is considerable literature suggesting that
intracellular calcium accumulation is a toxic process
involved in both focal and global neuronal injury
[72]. At least some of this calcium accumulation is
via classic membrane channels--both excitatory
neurotransmitter receptors (particularly NMDA
receptor) and voltage-dependent channels have
been implicated [6]. Voltage-dependent calcium
channel blockers such as Flunarizine have been
shown experimentally to confer some degree
of neuroprotection. However, while FIunarizine
clearly confers neuroprotection when given prior
to HI injury [44, 73-75], it has no effect given
immediately after injury [76] suggesting that acti-
vation of voltage-dependent calcium channels is
important in primary but not in secondary neuronal
death. The other major limitation of such blockers
is that they induce peripheral vasodilatation to a
greater or less extent, which inay aggravate
hypoperfusion during asphyxia [44]. Since cardio-
vascular compromise occurs at doses close to those
that confer a degree of neuroprotection, unaccept-
able hypotension led to the abandonment of early
clinical studies [77].
Anti-excitotoxic agents
Accumulation of excitatory amino acid (EAA)
neurotransmitters during hypoxia-ischaemia is well
documented [78]. The rise is related to failure of
energy-dependent re-uptake mechanisms and to a
lesser extent to hypoxia induced depolarization of
excitatory neurons [79]. The relative importance of
EAA exposure to primary injury in the developing
brain however remains controversial. Studies of
local injection of glutaminergic agents suggest that
there may be phases of relatively increased suscep-
tibility to excitotoxic injury during prenatal devel-
opment [80, 81]. Nevertheless, there is no clear
correspondence between regional levels of EAAs
during HI and subsequent injury [82]. Furthermore,
in vivo microdialysis studies in the fetal sheep
suggest that the primary rise in EAAs is relatively
modest and may be counterbalanced by a very
much larger rise in inhibitory transmitters, including
gamma amino butyric acid (GABA), during either
systemic asphyxia [83] or cerebral ischaemia [I1]. In
any event the toxicity of currently explored agents
makes neuroprophylaxis with antiexcitotoxins an
unlikely route for effective intervention [84, 85].
92
The possibility of rescue therapy with such
agents remains unclear. Post-asphyxial seizures
have been associated with disproportionate accu-
mulation of excitotoxins, with a fall in GABA [11].
This may in part be related to a greater sensitivity
to injury of inhibitory neurons in the primary phase
which may contribute to a subsequent loss of
suppression of excitatory neurons in the second-
ary phase [86, 87]. Both N-methyl-D-aspartate
(NMDA) and the non-NMDA [particularly alpha-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA)] type receptors have been implicated
in toxicity. Postasphyxial seizures can for example
be wholly blocked by the NMDA receptor antag-
onist, Dizocilpine (MK-801) [88]. While there is
some neuroprotective effect of MK-801 when
given after HI injury [88-90], it is far less effective
than when administered during the primary phase
[91-94]. Although the non-NMDA antagonists
have been proposed to be relatively more protec-
tive in the secondary phase, as discussed above
recent evidence suggests that much of the apparent
protection may have been mediated by a mild but
prolonged fall in systemic temperature [51].
Magnesium
MgSO 4 at high doses is a NMDA receptor
antagonist and in experimental paradigms reduces
excitotoxic injury [95]. It has been reported that
the use of MgSO 4 for tocolysis and the treatment
of preeclampsia is associated with a reduced inci-
dence of cerebral palsy [96]. However, recently
presented alternative analyses suggest the protec-
tive effect of the association with preeclampsia may
be independent of MgSO 4 [97]. Indeed, in prema-
ture neonates prenatal magnesium exposure does
not appear to ameliorate subsequent white matter
injury [98].
The levels of Mg 2+ reached after systemic
MgSO 4 therapy, as opposed to intracerebral injec-
tion [99] are not likely to cause significant inhibi-
tion of Ca 2+ conductances. In recent studies in the
fetal sheep [100], immature rat [101] and piglet
[102] no protective effect of either prophylactic or
rescue (post insult) MgSO 4 could be demonstrated.
The early multicentre clinical trial of magnesium in
perinatal asphyxia was thus based on very limited
evidence of its likely efficacy; it is unfortunate that
in the early phase of this trial high dose therapy
was associated with hypotension [103].
A . J . Gunn & P. D. Gluckman
Nitric oxide synthase (NOS) inhibitors
NO is a volatile, rapidly regulated gas which can
be produced by NO synthases in endothelial cells
(eNOS), neurons (nNOS) and by neutrophils or
microglia (inducible NOS, or iNOS). Because of
failure to take this into consideration the early trials
of non-selective NOS inhibitors produced contra-
dictory results; as discussed in detail below, the
available data suggest that prophylactic inhibi-
tion of nNOS in isolation may reduce neuronal
injury [104] while inhibition of eNOS is likely to
exacerbate it by impairing cerebral perfusion [105].
Endothelial NO is a vasodilator which under
physiological conditions plays an important role in
the regulation of CBF, cerebral autoregulation,
blood flow-metabolism coupling and the control of
platelet aggregation and adhesion [106, 107]. In
the primary phase many studies have shown that
during reperfusion there may be reduced NO
production and thus NO mediated cerebrovascular
vasodilatation is impaired [108-111]. There is evi-
dence to show in the adult that administration of
NO donors is cytoprotective and that general NO
inhibition exacerbates neuronal injury probably
by impairing perfusion [107, 108, 112]. NO inhibi-
tion also impairs cerebrovascular autoregulation
during moderate hypotension [113]. In the sheep
fetus it has been shown that NO plays a role in
maintaining normal fetal vascular tone and NO
appears to mediate the rises in CBF during hypoxia
[114]. Further, in the secondary phase, in vivo
microdialysis has demonstrated increased NO
synthesis which appears to mediate much of
the secondary hyperaemia [11]. Inhibition of NO
synthetase at this time leads to a reduction in
cerebral blood volume and to greater neuronal loss
[105, 1151.
Neuronal NO synthase (nNOS) expression in
the developing brain correlates with regions of
selective neuronal loss in the developing rat brain.
Specific inhibitors of nNOS during or immediately
after the primary phase have recently been shown
to improve neuronal outcome suggesting that this
component does contribute to reperfusion injury
[116, 117]. In vitro, NO also mediates some of the
cytotoxic actions of excitatory amino acids [118].
No studies reported to date have yet examined its
role in the secondary phase however.
Finally, iNOS, which is inducible by cytokines
and released by activated macrophages in very
highly concentrated killing bursts, may contribute
to reperfusion injury [1191. NO can combine with
Prevention of perinatal brain damage
superoxide anion to give rise to cytotoxic peroxy-
nitrite anion. Oligodendrocytes are also highly
sensitive to NO as compared to astrocytes and this
leads to necrotic death, thought to be a result of
mitochondrial damage [120].
Microglial activation
Microglial activation occurs early in severe injury
and later in mild injury. The activation is presum-
ably a response to tissue injury and altered surface
expression of major histocompatibility complex
(MHC) antigens on neurons or glial. Activated
microglia express a number of cytotoxic cytokines
such as tumour necrosis factor R (TNF-R) and also
the cytotoxic radicals NO and H20 2. Microglial
reactivity has probably evolved as a protective
response to viral and bacterial infection at the cost
of retaining a potential neurotoxic role [13]. Most
of the putative .inhibitors of microglial activation
(e.g. transforming growth factor [3) may have
alternate modes of inducing neuroprotection
[121, 1221.
Corticosteroids
In the neonatal 'Levine' rat model of hypoxia-
ischaemia, pretreatment with steroids has been
consistently shown to be protective [123], and to a
greater degree than with other modalities including
OFR antagonists and calcium channel antagonists
[124]. Although steroid treatment is associated
with hyperglycaemia, which is protective in the
neonatal rat, the treatment effect was greater than
seen with glucose infusions alone [125]. These
results should still be interpreted with some cau-
tion, as the data have not been extended to other
species or experimental approaches. Steroid pre-
treatment worsens outcome after ischaemia in adult
rats, because of associated hyperglycaemia [126].
The effect of maturation on the response to hyper-
glycaemia is related to the comparatively low
levels of neuronal glucose transporters in the
neonatal rat [127]. This is unlikely to be the case
in other species since hyperglycaemia during
hypoxia-ischaemia worsens outcome, for example,
in the piglet [128].
Despite these caveats, this approach remains of
particular interest since administration of dexa-
methasone in premature labour improved neuro-
logical function in surviving infants [129]. Clearly
93
this beneficial outcome might also be related to
other systemic effects such as improved pulmonary
function, and so cannot be clearly attributed to a
neuroprotective effect, but it is a strong induce-
ment to extend experimental investigation of this
approach.
Endogenous protective
mechanisms
In devising neuroprotective strategies it is import-
ant to consider what endogenous mechanisms the
CNS itself utilizes to restrict injury. At least four
endogenous protective mechanisms exist: neuro-
modulators; neurotrophins; cerebrovascular adapta-
tions; and cellular factors. A number of possible
interventions have been proposedi primarily based
on the first two factors.
Inhibitory neuromodulators
First, inhibitory neuromodulators such as GABA
and adenosine may partially antagonize the neural
effects of the EEAs [17]. Microdialysis experiments
in the fetal sheep suggest that this endogenous
response is greatest during the primary phase,
while the endogenous post insult elevation is likely
to be limited to the reperfusion phase, and early
part of the latent period [11]. Adult species such as
the turtle that are very tolerant to hypoxia, show a
similarly elevated GABA response to anoxia, which
is suggested to reduce cerebral energy consump-
tion [130]. Consistent with this hypothesis,
GABAergic agonists have been shown to have
neuroprotective properties during (but not as yet
after) ischaemia, both alone and in combination
with NMDA antagonists [131, 132].
There is evidence that endogenous adenosine
has a significant role in the brain after hypoxia-
ischaemia, since theophylline, an adenosine antag-
onist, worsens delayed neuronal death when
given post insult [133, 134]. Pre-treatment with
long acting analogues of adenosine, or upregula-
tion of adenosine receptors has been reported to
reduce neuronal loss, in some [133, 135, 136] but
not all studies [17]. Rescue therapy however has
not been consistently effective, and to date the
cardiovascular side-effects remain unacceptable
[1371.
94
Anti-apoptotic therapy--endogenous
growth factors
While the observation that the brain enhances
neurotrophic activity (defined by the ability to
support neuronal survival in vitro) after injury is of
long standing [19], the neurotrophins involved
have only recently been identified. Extensive
studies have been performed after unilateral HI
injury in the immature rat. There is minimal induc-
tion, shortly after the end of hypoxia, of mRNAs
coding for members of the nerve growth factor
family [NGF]3, brain-derived neurotrophic factor
(BDNF), neurotrophin 3 (NT3)]. Such induction
is restricted to the hippocampus of the non-
injured side and has been shown to be induced by
postasphyxial seizures [138].
Broader spectrum growth factors however are
markedly induced by HI. Most attention has
focused on insulin-like growth factor 1 (IGF-1)
which has been shown to block developmental
apoptosis in motoneurons in vivo [139], and exper-
imental apoptosis in cerebellar granule cells in vitro
[140]. After injury, IGF-1 mRNA is induced in
injured glia ~n a dose-related manner 3-5 days after
injury [141]. IGF-1 protein can be shown to be
largely associated with astrocytes at 3 days after
injury [141]. Similarly, after electrolytic damage,
enhanced IGF-1 release has been found in micro-
dialysate several days later [142]. Two of the IGF
binding proteins (IGFBP), IGFBP-2 and IGFBP-3 are
also induced early after injury [143, 144]. In con-
trast, IGF-2 and IGFBP-5 are not induced until
3-10 days after injury, presumably as part of
wound repair mechanisms [145, 146].
Basic fibroblast growth factor (bFGF) is also
induced after injury but in a more limited manner
and also has neuroprotective effects. There is one
report suggesting its actions are mediated by IGF-1
induction [147]. Transforming growth factor ~, and
activin are two members of a large evolutionarily
related family of growth factors and are induced
after injury. TGF-~I is neuroprotective but it is not
clear whether it is acting as atrophic factor or to
inhibit microglial activation [121, 122].
IGF-1
IGF-1 has recently been shown to have significant
potential as a neuronal rescue agent [148], presum-
ably by inhibiting apoptosis. The rationale was that
the IGF-1 system was very specifically induced by
A . J . Gunn & P. D. Gluckman
injury and IGF-1 is a potent and broadly active
neurotrophic agent in vitro, in contrast to the NGF
family whose actions are limited to specific cell
types.
In adult rats subject to HI, IGF-1 given as a
single dose i.c.v. 2 h after injury reduced both
infarction and selective neuronal loss in a dose-
dependent manner [148]. Neurodevelopmental out-
come was also improved. In contrast IGF-1 given
prior to injury was not neuroprotective. Studies
with [3H]IGF-1 show that it is rapidly transported
via the perivascular space to the injured regions
[149]--this localization may be a result of early
induction of IGFBP-2 [144]. In late gestation fetal
sheep subject to severe global ischaemia, IGF-1
given as a single dose in to the lateral ventricle 2 h
after reperfusion reduced neuronal loss, reduced the
secondary rise in cytotoxic oedema, reduced the
incidence of postasphyxial seizures and lowered
the magnitude of lactate production [150]. No
systemic effects were evident within the therapeu-
tic range, and IGF-1 was effective at a very low
dose (0.1-1 Lug i.c.v, to a 3-kg fetus).
The actions of IGF-1 may be mediated in part by
derivative molecules. In neural tissue a protease
cleaves IGF-1 at its N-terminal to produce a
tripeptide, glycine-proline-glutamate (GPE), and
des (1-3) IGF-1. des (1-3) IGF-1 is fully active at
the IGF-1 receptor but has low affinity for IGFBPs;
it has also been shown to be neuroprotective at
correspondingly higher doses, confirming that pro-
tection is mediated through the IGF-1 receptor
[151]. Interestingly, at equimolar doses to IGF-1,
GPE is also significantly protective, although to a
lesser extent. The receptor system mediating the
actions of GPE is unclear--it may be a glutamate
receptor subtype but the protective action was not
mimicked by treatment with the highly selective
NMDA antagonist, MK-801 (unpublished data). In
summary, it is likely that IGF-1 is transported to
the region of injury in association with IGFBPs,
then is proteolytically cleaved to des IGF-1 which
is anti-apoptotic and to GPE which acts as a
neuromodulator--thus IGF-1 may be a key endog-
enous neuronal rescue system. Figure 3 shows
schematically the proposed sequence of actions of
IGF-1.
Selection and monitoring of patients
A key issue is how patients will be selected for
neuronal rescue therapy. Only a minority of
Prevention of perinatal brain damage 95

IGF-1 + IGFBP
Ventricle
Complex

ProteaseBp

Damaged IGF-1 + IGFBP

tissue

=. . . . . . l ......................
f ProteasemF
I
• v
i/
IGF-1 IGF-1
~~ 1 ~ I ~
? I~A--. ?

j ".d e s - I G F - l ' " ~ " ' " ~ GPE

v ~'" ",g f

Figure 3. This diagram shows the known (filled arrows) and potential (dotted arrows) neurotrophic influences of IGF-1.
Proteases cleave IGFBPs to release IGF-1 which can interact with receptors (R1) on neurons (NA). Glia (GA) have receptors
(RI) which respond to IGF-1 and produce neurotrophins (NTY) which may interact with neuronal receptors (RY). GPE may
be synthesized from IGF-I by protease cleavage and appears to bind to a neuronal (NB) receptor (R2) or binding protein. It
may also interact with a receptor (R2) on glia (GB), stimulating other neurotrophin (NTX) production and receptor (RX)
interactions. N-: Neuron; G =Glia; R1 = IGF-1 receptor; R2 = GPE receptor; NTX,Y= Neurotrophin; RX,Y= Neurotrophin
receptor; Protease BP is specific for IGFBPs; Protease IGF truncates IGFs at the N-terminal tripeptide to produce GPE.

patients resuscitated from an asphyxial episode
(particularly birth asphyxia) are destined to develop
a secondary phase of injury which can be manipu-
lated. Yet it is only in that group that intervention
would be useful. Studies in animals suggest that
a variety of biophysical measures (cortical im-
pedance, electrophysiology, magnetic resonance
spectroscopy, near infra-red spectroscopy) can be
utilized in the latent phase to predict outcome [14,
24, 33]. Cortical impedance and electrophysiology
are low cost technologies which can be employed
at the bedside. At present however, the earliest that
infants with a poor prognosis can be reliably
identified is approximately 6 h after birth [152].
Other possible approaches may include biochemi-
cal indices such as CSF lactate [153], or positron
emission tomography evaluation of cerebral metab-
olism after resuscitation [154]. Active clinical
research in these areas is needed and will be an
essential prerequisite to logical introduction of
neuronal rescue therapies.
Conclusions
The development of a rational approach to clinical
intervention in HIE depends on understanding the
multiple mechanisms involved and their temporal
relationship. Neuronal rescue and neuroprophylaxis
are highly likely to require distinct therapeutic
approaches. A range of strategies have been devel-
oped that are potentially applicable to clinical
96
practice. While these experimental studies are very
promising, a cautious approach to clinical exploita-
tion is still needed, as reviewed elsewhere [155].
We can be optimistic however that the knowledge
gained over the last decade will ultimately lead to
effective therapies.
Acknowledgements
The authors' work reviewed in this chapter is funded by
grants from the Health Research Council of New Zealand and
the National Institutes of Health HD 32752.
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