Pharmacological strategies for the prevention of perinatal brain damage

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 pharmacological 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 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 understanding 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 interventions. 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 hypotension [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

mechanisms are hypoxic depolarization and accumulation 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 intracellular calcium exposure, may trigger secondary pathologic processes leading to delayed to secondary 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 cytotoxic 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 systems 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

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may be related to much greater accumulation of inhibitory neurotransmitters during asphyxia [11], and of neurotrophic factors [19] after injury.

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The timing of cell death Although some neurons and glia die during the primary phase, during or shortly after the asphyxial insult, the seminal observation in the last decade is that many cells at least partially recover but go on to die many hours later. The longer and more severe the insult, the greater the proportion of primary neural injury [10, 21]. There is now good experimental evidence for the occurrence of delayed neuronal death both in immature and adult animals [21-24] and after cardiac arrest in man [25]. Consistent with these histological data, a secondary phase of deterioration has been clearly demonstrated clinically in birth asphyxia. Some asphyxiated infants have normal cerebral energy metabolism shortly after birth as measured by magnetic resonance spectroscopy (MRS), but then show secondary failure of energy metabolism many hours after birth, which reaches a maximum by 48 h [26]. Subsequently, neuro-developmental outcome correlates with the degree of energy failure [27]. Further supportive evidence for a significant phase of secondary injury in man comes from measurements of cytochrome oxidase [28], the development of cytotoxic oedema in neonatal encephalopathy [29], and from electroencephalographic studies which confirm the delayed evolution of seizure activity after asphyxia [30]. Experimentally, a similar pattern has been shown in studies of severe hypoxia-ischaemia in the piglet [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

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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

89

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Types of cell death Two morphological patterns of cell death are seen--necrosis and apoptosis [21, 34, 35]. Typically, 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 hypoxicischaemic 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 development [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 demonstrated 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

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: 'neuroprophylaxis' 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 inhibitory neurotransmitters/neuromodulators and antiapoptotic therapy such as growth factors. Figure 2 illustrates the effect of selected putative prophylactic 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

A . J . Gunn & P. D. Gluckman

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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

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 between drug induced vasodilatation and asphyxial cardiac injury, which may cause further hypotension and secondary cerebral compromise [44]. One other major issue to consider when evaluating studies is temperature. It is well known that small changes in temperature, of only a few degrees, during and immediately after hypoxiaischaemia, can critically modulate damage [45]. This confounded early studies of antiexcitotoxic therapy; the apparent benefits of NMDA antagonists seem to have been either partly due to associated 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 protective [49, 50], and that this can be produced, for example, by anti-excitotoxic agents [51]. The reciprocal issue arises with mild hyperthermia during the secondary phase, which occurs in several species after stroke; unless this is controlled, 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 hypoxiaischaemia [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 electron chain, as well as other oxidation-reduction reactions in the cytoplasm normally produce oxygen 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 degradation 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 relationship 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 extracellular calcium has been shown to uncouple mitochondrial electron transport, and so to promote free radical production [58]. Increased production of oxygen free radicals has been implicated during asphyxia, and more particularly 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 during 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 deferoxamine, 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 ganglioside 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 neuroprophylaxis [69, 70].

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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 activation 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 cardiovascular compromise occurs at doses close to those that confer a degree of neuroprotection, unacceptable 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 susceptibility to excitotoxic injury during prenatal development [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].

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The possibility of rescue therapy with such agents remains unclear. Post-asphyxial seizures have been associated with disproportionate accumulation 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 secondary phase [86, 87]. Both N-methyl-D-aspartate (NMDA) and the non-NMDA [particularly alphaamino-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 antagonist, 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 protective 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 incidence of cerebral palsy [96]. However, recently presented alternative analyses suggest the protective effect of the association with preeclampsia may be independent of MgSO 4 [97]. Indeed, in premature 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 injection [99] are not likely to cause significant inhibition 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 contradictory results; as discussed in detail below, the available data suggest that prophylactic inhibition 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 evidence 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 inhibition 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 peroxynitrite 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].

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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 inducement to extend experimental investigation of this approach.

Microglial activation Microglial activation occurs early in severe injury and later in mild injury. The activation is presumably 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 hypoxiaischaemia, 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 caution, as the data have not been extended to other species or experimental approaches. Steroid pretreatment worsens outcome after ischaemia in adult rats, because of associated hyperglycaemia [126]. The effect of maturation on the response to hyperglycaemia 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 dexamethasone in premature labour improved neurological function in surviving infants [129]. Clearly

Endogenous protective mechanisms In devising neuroprotective strategies it is important to consider what endogenous mechanisms the CNS itself utilizes to restrict injury. At least four endogenous protective mechanisms exist: neuromodulators; neurotrophins; cerebrovascular adaptations; 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 consumption [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 hypoxiaischaemia, since theophylline, an adenosine antagonist, worsens delayed neuronal death when given post insult [133, 134]. Pre-treatment with long acting analogues of adenosine, or upregulation 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.

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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 induction, 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 noninjured 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 experimental 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 microdialysate 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 contrast, 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].

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 dosedependent manner [148]. Neurodevelopmental outcome 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 therapeutic 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 protection 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 endogenous neuronal rescue system. Figure 3 shows schematically the proposed sequence of actions of IGF-1.

IGF-1 IGF-1 has recently been shown to have significant potential as a neuronal rescue agent [148], presumably by inhibiting apoptosis. The rationale was that the IGF-1 system was very specifically induced by

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

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patients resuscitated from an asphyxial episode (particularly birth asphyxia) are destined to develop a secondary phase of injury which can be manipulated. Yet it is only in that group that intervention would be useful. Studies in animals suggest that a variety of biophysical measures (cortical impedance, 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 biochemical indices such as CSF lactate [153], or positron

emission tomography evaluation of cerebral metabolism 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 developed that are potentially applicable to clinical

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practice. While these experimental studies are very promising, a cautious approach to clinical exploitation 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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