Neonatal Hypoxia Ischaemia: Mechanisms, Models, and Therapeutic Challenges

1The clinical definition of neonatal HI injury is “asphyxia of the umbilical blood supply to the human fetus occurring at 36 gestational weeks or later”. Neonatal HI is synonymous with hypoxic-ischaemic encephalopathy (HIE) occurring in the term infant, where term is defined as 36 gestational weeks or later. This review addresses neonatal HI or HIE, any results concerning perinatal hypoxic-ischaemia injury will be clearly indicated in the text. This disorder encompasses a large range of physiological origins and clinical outcomes. The diagnostic criteria for neonatal HI are based on a set of markers demonstrated to correlate with clinical outcome. These include: 5-min Apgar score of less than 5; need for delivery room intubation or CPR; umbilical cord arterial pH less than 7.00; and abnormal neurological signs, such as hypotonic muscles or lack of sucking reflex. Electroencephalography (EEG) has also proved helpful as a predictor of clinical outcome (reviewed in; ). Amplitude-integrated EEG (aEEG) in particular, a filtered time-compressed continuous one- or two-channel read-out, has been demonstrated a reliable predictor in meta-analyses up to 5 years after birth, however, some report that aEEG remains less reliable than MRI, especially following hypothermia treatment. The Thompson score, an EEG measure of predictive neurodevelopment, is likely to remain useful to clinicians. This is by no means an exhaustive list of risk factors and signs of clinical concern that can occur during the early postnatal period.

2Neonatal HI is the most common cause of death and disability in human neonates, accounting for 23% of infant mortality worldwide, and affecting 0.7–1.2 million infants annually (; see Figure 1). In developed countries, incidence of HI injury has not decreased in the past two decades, remaining a significant cause of fatality and disability. The frequency of motor and cognitive disorders linked to perinatal and early postnatal brain injury actually increased during the 1990s, and currently remains stable. Progress in assisted respiratory and intensive care technology has led to greater than 90% survival of infants born after gestational week 23, perhaps accounting for the increased burden of disability within the population as mortality rates have decreased substantially.

3Amongst those who survive the initial injury, rates of disability remain high throughout life. Of patients surviving neonatal HI, 5–10% of infants demonstrate persistent motor deficits, and 20–50% display sensory or cognitive abnormalities that persist to adolescence. A meta-analysis of seven studies including 386 infant patients investigated the average incidence of mortality and morbidity: 5.9% of all patients across all studies died, 16.3% suffered neonatal seizures, and 17.2% experienced neurological deficits, with 14.2% qualifying for a diagnosis of cerebral palsy. Long-term outcomes in neonatal asphyxia infants have also been investigated. One meta-analysis found that 1–18% of patients were identified as having severe sensorimotor or learning disorders by the age of 2–5 years, with only 50–60% of patients reported as developmentally normal. This study covered a wide range of injury severities and follow-up ages. Disorders included seizures, hearing and vision loss, language disorders, microcephaly, and muscle spasticity. Studies also report more severe neurological signs in patients suffering severe HI compared to those with milder HI injury. Yet, many individuals who showed abnormal neurological signs at birth were normal at 2-year follow-up. Clinical features and outcomes of neonatal HI are summarized in Figure 1.

4Despite the high disability burden associated with surviving neonatal HI patients, there are very few preventative or protective treatments available for infants suspected to have suffered an HI event. The only licensed treatment currently available is hypothermia. This treatment involves subjecting either the infant’s whole body or head-only to temperatures of around 33°C. Hypothermia was first demonstrated to improve survival in cases of cardiac arrest, and has since been applied as a neuroprotective treatment in acute neonatal HI injury patients. A recent meta-analysis found that hypothermia carried out in over 1,200 infants reduced the rate of death and neurological handicaps at 18 months follow-up across all severity categories of neonatal HI injury. However, hypothermia alone is not sufficient to prevent all brain injury or neurological symptoms, highlighting the need for additional therapies to use in conjunction. Xenon gas administration is currently being trialed as an additive therapy alongside hypothermia. There are currently no other licensed treatments available for neonatal HI.

5Despite the efficiency of hypothermia, death and disability remain a common feature of neonatal HI prognosis. Observations concerning global prevalence and poor long-term outcome reiterate the urgency of finding novel neuroprotective treatments for use during and directly following HI injury. This review examines the neuropathology resulting from neonatal HI injury in humans. The review then examines currently available animal models of neonatal HI and summarizes the strengths and weakness of such models for research into this complex human condition. Finally, the review will detail a potential approach toward identifying new pharmacological targets for neonatal HI therapies, focusing on the protein neuroserpin.

6There is ample evidence that brain damage occurs in human neonatal HI patients with poor clinical outcomes, documented in both imaging and histopathological studies. Neuropathology has been characterized in human post-mortem studies, concluding that most areas of the brain are vulnerable to some extent to neonatal HI injury. Gray and white matter lesions have been described at term after HI. Localization and extent of neuropathology has been shown to be associated with neurodevelopmental symptoms, giving insights into the nature of disability the patients may present with.

7Infants who survive the initial HI insult display cerebral damage visible with structural imaging. Magnetic resonance imagining (MRI) studies of term infants with neurological signs and combinations of fetal distress, cord acidemia, and depressed Apgar scores have been carried out in over 1,000 infants (reviewed in ). These studies report great variation in the anatomical areas involved between individual patients, yet most samples described either patients demonstrating predominant or substantial injury to cerebral cortex, or basal ganglia and thalamus in partially overlapping subpopulations. These two patterns of injury are shown in Figure 2. Cerebral white matter has been described as selectively sensitive to term HI injury. Although less common, severe selective involvement of subcortical white matter has been documented. One review described the literature concerning structural MRI scans in the acute phase (within 2 weeks after birth): approximately 15–30% of scans were normal, lesions in basal ganglia and thalamus are present in 40–80% of cases, with abnormalities of watershed white matter and cortex present in 40–60% of patients. MRI anatomy has been shown to agree well with post-mortem studies. Therefore, no single area of the brain is specifically damaged following neonatal HI. Any future treatments should take this diversity into account and provide neuroprotection to neurons throughout the brain.

8The MRI scan is currently the method of choice for investigation of neonatal anatomy in both clinical and experimental circumstances. Diffusion-weighted MRI imaging has greatly improved identification of the time of onset of brain lesions. A reduced diffusion coefficient can be calculated, showing restricted diffusion during the first few days after the insult, with pseudonormalization by the end of the first week. Sequential imaging has shown that lesions in the basal ganglia may increase in size during the first week after birth, and asymmetric diffusion within white matter has been correlated with clinical severity of hemiparesis. Cranial ultrasound also remains a valuable clinical tool. Another technique promising to add to understanding of neonatal HI is magnetic resonance spectroscopy (MRS), which allows brain metabolism to be imaged in real time. Full-term neonates with perinatal asphyxia have been studied, indicating that brain metabolism becomes abnormal after 6 to 12 h only to decrease even further after 24 h. This coincided with clinical deterioration such as development of seizures. The concept of a delayed metabolic abnormality or ‘secondary energy failure’ has been elaborated in animal models. Using MRS in these animal models, neuroprotective strategies could be tested. By using MRS data as real-time measurements of decreased brain metabolism in brain injury. 1H-MRS and 31P-MRS studied have demonstrated metabolic abnormalities following HI insult, which may persist for weeks. Unfortunately, with the magnetic field strength of current clinical systems, only large brain areas can be examined, limiting the use of this technique. Infants who have suffered neonatal HI often exhibit abnormal EEG activity (reviewed in; ). A range of abnormalities have been described, including: low voltage in isoelectric EEG, mild voltage depression, and asymmetry of trace, although all of these criteria have been differently defined by different analysts. However, imaging techniques are constantly improving.

9Lesions occur in many clinical patients, yet the effect on cognitive function is as diverse as the neuroanatomy. The area of cortex and basal ganglia damaged during the initial HI injury is directly predictive of language and motor outcome in childhood. Examination of diffusion-tensor imaging of neonates was predictive of survival and motor outcome. Children with basal-ganglia-thalamus pattern of injury tend to be severely disabled due to dyskinetic cerebral palsy and epilepsy. Infants with predominant watershed white matter and cortex lesions have more prominent cognitive than motor deficits. Severe motor impairment is uncommon, and this group is often considered to have a normal outcome when seen at 12–18 months, although suboptimal head growth, behavioral problems, epilepsy, and a delay in language emerge during late childhood. Therefore, cortical damage appears to be relevant to functional outcome in surviving neonatal HI patients. This information could be used to predict future susceptibility to disability before it manifests, allowing social and educational supports to be put in place early.

10Anatomical studies describe loss of brain volume following moderate and severe neonatal HI. However, the underlying molecular mechanisms responsible for cell death are debated. Many pathways have been implicated in HI injury in the term brain, primarily: excitotoxicity, oxidative stress, and inflammation. Molecular studies have drawn attention to a fact essential for the development of successful new therapies that the neonatal brain and its injury is fundamentally different from that seen in adult HI stroke injury.

11There are many important differences between neonatal HI and adult ischaemic stroke. For example, severe HI events in the infant brain can lead to liquifactive disintegration, not seen after adult stroke. Newly formed blood vessels are fragile and prone to rupture, and surrounded by fewer astrocyte end-feet. Another key site of difference is the BBB. Studies in rodents indicate that the BBB is compromised as a result of neonatal HI. Yet the common belief that the neonate BBB is less effective has recently come under revision. Tight junctions, the occlusive element of the BBB, are present as soon as embryonic vessels invade the brain, and are functional. In a model of hypoxia in newborn piglet, BBB integrity was maintained, yet other experiments have demonstrated damage to the BBB following neonatal HI.

12Cerebrovascular autoregulation is another factor which must be considered in neonates. The concept that preterm infants have a ‘pressure passive’ cerebral circulation is widely accepted. However, sick term infants demonstrate impaired autoregulation and the range of blood pressure over which cerebrovascular autoregulation functions expands with maturity. Also, the concentrations and actions of various signaling molecules is different in the developing brain including; caspase-3, VEGF, and HIF-1, among others (reviewed in ).

13One surprising difference is sexual dimorphism in response to neonatal HI. Male babies are at higher risk of cerebral palsy than females. Cognitive and motor outcomes are worse in male than in female low birth weight infants. Quantitative imaging shows that male premature infants are more vulnerable to white matter injury, whereas females are more vulnerable to gray matter injury. This sex difference has also been replicated in rodent in vitro models of hypoxic cell death. Although many molecular mechanisms are currently under investigation, this sexual dimorphism remains largely unexplained. Therefore, the unique state of the developmental brain should always be at the forefront of the researcher’s minds.

14Neonatal HI injury evolves over time. Injuries seen with MRI scans within the first few hours after asphyxia are subtle, restricted diffusion typically starting as small lesions in the putamen and thalami, progressing over the next 3 to 4 days to involve more extensive areas of the brain. Within the first few hours, regionally specific fluctuations in blood flow trigger excitotoxicity, free radical generation, and edema. A secondary phase of injury occurs during the following hours and days, resulting in neuroinflammation, mitochondrial permeabilization, and loss of cerebral autoregulation. A tertiary phase of brain injury has been proposed, which may exacerbate injury through persistent inflammation.

15The balance between molecular cell-death processes which cause this damage in neonatal HI remains debated. Early evidence indicates that the majority of cell death in neonatal HI is necrotic, however, all regions also undergo increased apoptotic death. Some studies suggest a more prominent role for apoptosis. Immature neurons in vitro are more susceptible to apoptotic death than mature neurons. Others report that necrosis is the major cellular pathology in humans and animals. Yet others recognize that both occur. Some report that necrosis predominates in severe cases, whereas apoptosis occurs in milder injury. Neurons often display morphologic features along an apoptosis-necrosis continuum. In addition to apoptosis and necrosis, some neurons in the neonatal HI brain undergo autophagy (reviewed in;; ). Neuronal autophagy occurs in rodent neonatal HI models. However, there is conflicting evidence as to whether the occurrence of autophagy augments brain damage, or prevents the spread of necrotic cell death. Artificially exclusive classification of cell death may hinder research and therapy development.

16To add to this complexity, neonatal HI injury appears to activate several interacting molecular cascades. A simple schematic of the three major cascades is shown in Figure 3. The first is excitotoxicity, through which physiological glutamate neurotransmission leads to overactivation of postsynaptic receptors and cell death (reviewed in;,;; ). The N-methyl-D-aspartate (NMDA) receptor is relatively over-expressed in the developing brain. In P6 rats, the NMDA receptor is expressed at 150–200% of adult levels. The predominating combination of NMDA receptor subunits in the perinatal period seems to favor prolonged calcium influx for a given excitation. The same NMDA receptor that promotes plasticity can lead to massive Na+ and water influx, cellular swelling, pathologically elevated intracellular calcium, and energy failure, leading to a ‘spiral of death’. Oxygen glucose deprivation (OGD) in rat hippocampal neurons leads to a marked reduction in glutamate removal from the synapse. Injection of NMDA into rat brain produces more extensive cell death in the neonate than in the adult. Elevated glutamate has been documented in the cerebrospinal fluid (CSF) of infants who have suffered severe HI injury. The neonatal brain is much more prone to seizure activity than the mature brain, suggesting a prominent role for neuronal hyperexcitability and excitotoxicity although the molecular mechanisms behind this have not been fully elucidated (reviewed in ). However, seizure activity could also be explained by paradoxical excitatory activity of the neurotransmitter gamma-amino butyric acid (GABA) in the developing brain. Drugs that block NMDA receptors are protective against HI injury in neonatal rodent models. Activation of AMPA receptors also contribute to injury, however, AMPA antagonists are not as protective. These findings are yet to be exploited in human clinical trials, as the integral role of glutamate receptors in healthy neuronal plasticity could be damaged by the use of NMDA and AMPA antagonists at such a sensitive developmental stage.

17An integrally linked cascade is that of oxidative stress. Excitotoxicity causes energy depletion, mitochondrial dysfunction, and cytosolic calcium accumulation, which in turn leads to generation of free radicals. Free radicals alter membrane pump function, allowing more glutamate release and NMDA receptor activation, leading to more excitotoxicity. Oxidative stress is a general term for the increase in free radical production as a result of oxidative metabolism under pathologic conditions. When oxygen floods the microenvironment of cells damaged by hypoxia, mitochondrial oxidative phosphorylation is overwhelmed and reactive oxygen species accumulate. Fetal life elapses in a low oxygen environment. In the first minutes of life, an abrupt increase in O2 partial pressure occurs, which creates a pro-oxidant condition. During birth asphyxia, excess calcium influx and other factors lead to severe oxidative stress. There is accumulation of hydrogen peroxide after HI in neonatal mice but not in adults. Because of its high lipid content, the brain is particularly susceptible to free radical attack. The polyunsaturated fatty acid content of the brain increases during gestation. Lipid peroxidation may be a major factor in the white matter damage. The developing brain’s immature antioxidant defense also contributes to sensitivity to oxidative stress. Adequate stores of antioxidants are necessary to protect against oxidative injury. Specifically, depletion of neuronal reduced glutathione exacerbates oxidative injury.

18Finally, inflammation is a major component of neonatal HI injury. Low-dose treatment with intrauterine LPS dramatically increases severity of HI injury in neonatal mice, but protects against HI in adult rodents. Intracerebral injection of NMDA receptor agonist produces a pattern of white matter injury, in which pretreatment with systemic IL-1ß, IL-6, IL-9, or TNF-α leads to a significant increase in lesion size. There is now substantial experimental evidence that intrauterine inflammation can exacerbate neonatal HI, which some have referred to as the “double-hit hypothesis” (reviewed in;; ). Microglia, the resident macrophages of the CNS, are among the first cells to become activated after HI. Activated microglia migrate to damaged regions and produce inflammatory cytokines, glutamate, nitric oxide, and free radicals. Drugs that block microglial activation protect the neonatal brain. Following hypoxia-ischemia, compromise of the BBB allows the entry of macrophages. Astrocytes also play a role in inflammation. CSF cytokines are elevated in term infants who later develop cerebral palsy. The diverse network of interacting mechanisms demonstrate the molecular complexity of neonatal HI injury. Potential protective treatments should strive to tackle common mediators of these cascades relevant to all three pathways, otherwise full protection will not be achievable.

19Neonatal hypoxia ischaemia has been modeled extensively in mice and rats (reviewed in;;; ), with a minority of researchers also studying larger animals such as pigs, sheep (reviewed in; ) or primates. Models intending to replicate the clinical symptoms of neonatal human HI can be roughly divided into the four categories discussed below. All models have distinct advantages and disadvantages. A summary of available rodent models is shown in Figure 4.

20Most published studies modeling neonatal HI in animals have employed the Rice–Vannucci model. This model comprises unilateral carotid artery ligation, recovery with the dam for approximately 1 h, followed by exposure to 8% oxygen for 1–3 h at 37°C. Although, the model was initially described in rat, it has been successfully adapted for mouse with similar anatomical and behavioral effects.

21Rice’ and Vannucci’s model replicates anatomical damage seen in human neonates. Their initial study showed selective gray-matter sensitivity to neuronal necrosis, with gray matter injury observed in cortex, hippocampus, thalamus, and basal ganglia, encompassing the sites damaged in human neonatal HI. Histologically, there is a gradation of injury that correlates with the duration or severity of insult. White matter lesions have also been described in this model, the extent of which correlate with the duration of exposure to hypoxia. However, bilateral common carotid artery ligation appears a stronger model of white matter damage. Metabolic alterations in the Rice–Vannucci model include decreased cerebral blood flow, brain acidosis, and decreased cerebral glucose uptake. An inflammatory response has also been demonstrated.

22Another convincing aspect of the Rice–Vannucci model is its ability to predict the therapeutic effect of hypothermia following the neonatal HI event. Mice treated with hypothermia showed smaller lesion volumes, in addition to better performance on the Morris water maze and circling tests. Many papers have investigated the behavioral outcomes of Rice–Vannucci injury in adult rodents. This model gives rise to well documented behavioral phenotypes including: impaired spatial learning and memory; impaired motor function as assessed by rotarod test, open field and motor reflexes; sensory processing abnormalities; and other cognitive phenotypes, such as reduced attention. Despite this broad range of documented effects, there are some contradictions between individual investigators (reviewed in ), which suggest that different genetic backgrounds, severity, or experimenters can significantly affect the outcome of Rice–Vannucci model.

23The Rice–Vannucci model of neonatal hypoxia ischaemia has several advantages. One is its prevalence, allowing direct comparisons with many other published papers. Another is that the contralateral hemisphere, exposed to hypoxia in the absence of ischemia, appears normal, providing a control hemisphere within the experimental brain. Thorough behavioral characterization support the long term consequences of this model mimicking neonatal HI. One significant drawback of this model is the high variability in size and severity of infarct between animals, making comparisons between experimenters difficult. Additionally, the invasive nature of severing the common carotid artery does not replicate human injury; such severe vascular abnormalities occur rarely, if at all.

24Some experimenters induce hypoxia in rodents exclusively using an oxygen deprivation chamber, without a preceding ischaemic procedure. These models are not as widely used as the Rice–Vannucci method, but have the potential to describe milder injuries and avoid the unphysiological occlusion of the common carotid artery. There are currently no published reviews or meta-analyses of hypoxia-only investigations, Supplementary Table 1 contains a brief summary of 122 published papers using hypoxia-only methodology compiled from Pubmed search results.

25Historically, these methods have been used to investigate hypoxic brain biochemistry. Several studies have documented altered levels of neurotransmitters. However, there is little consensus over the direction or magnitude of changes. Hypoxia-only models have become an established model used to generate seizures in neonatal rats. In one such model, P6 rat pups were placed in chambers at 9% O2 partial pressure and 20% CO2 partial pressure for 60 min. Some pups were immediately restored to room air, whereas others underwent gradual reduction of CO2. Pups which underwent hypoxia with immediate restoration of CO2 had a greater mortality rate and higher seizure frequency. However, subsequent anatomical analysis of these brains at P8 (; Wang et al., unpublished), failed to show any differences in expression of cell death markers or layer-specific markers of healthy cortical neurons. Therefore, the hypoxia-only insult resulting in seizures appears to generate only subtle injury to the brain, far short of that seen in some human patients.

26A range of behavioral phenotypes have been reported in hypoxia-only models, including hyperactivity; increased aggression, altered ultrasonic vocalization, and disturbed sleep. Relatively few investigators have pursued standard tests of spatial memory and locomotor behavior, and have obtained mixed results. The majority of hypoxia-only phenotypes are based on custom behavioral tests, making them difficult to compare to other established animal models. Additionally, some studies report no behavioral deficit following neonatal hypoxia alone.

27Although hypoxia-only models offer the potential to replicate the mechanism of hypoxia without major ischaemia seen in human neonatal HI patients, the models currently available are not ideal. One significant problem is the lack of methodological unity between different experimenters. There is little consensus on age of animal, background strain, oxygen partial pressure, time of exposure to hypoxia, or body temperature (see Supplementary Table 1). One example demonstrating the relevance of close control of these variables is temperature. P0 rat pups exposed to anoxia exhibit behavioral defects when anoxia was conducted at 39°C, yet not at 33 and 36°C (,;; reviewed in ). Therefore, far more care is needed to justify the design of hypoxia-only experiments before these models can be considered dependable models of human neonatal HI.

28Intrauterine infection is strongly associated with preterm birth and brain injury. Many models have been described which introduce different inflammation-inducing molecules at different ages, many of which cause cerebral inflammation and white matter damage seen in human patients.

29Administration of live E. coli into the uterus of pregnant rats can result in neutrophil infiltration in the fetal brain, increased fetal reabsorption and stillbirth, while surviving pups exhibit increased brain chemokines, cytokines, white matter injury, and behavioral phenotypes. The effects of bacterial mimetics such as the cell wall component lipopolysaccharide (LPS), have also been investigated. Intracervical injection in embryonic day 15 (E15) mice was associated with mild white matter injury but no behavioral deficits, whereas repeated intracervical LPS was associated with delayed neurosensory development. Other inflammatory models include viral infection simulated by injection of poly(I:C), a synthetic double stranded viral RNA, injection of which is associated with long-term behavioral deficits. Postnatal administration of inflammatory agents is widely used in rodents to model postnatal infection. Subcutaneous injection of live E. coli to P3 mouse pups was associated with microgliosis, loss of oligodendrocytes, and impaired motor coordination. However, most techniques which employ live bacterial injection have very high mortality rates. Postnatal intraperitoneal injection of LPS can also cause white matter damage and cerebral cytokine response. Repeated daily injection of LPS in mice resulted in elevated serum IL-6, reduced gray matter volume, decreased oligodendrocyte numbers, and decreased myelin staining. Similarly, repeated IL-1β injection in P1–P5 mice has been associated with impaired oligodendrocyte progenitor maturation, and severe memory deficits.

30One of the strengths of the inflammation model is that it reflects the exposure to infectious or inflammatory agents present outside of the highly sterile individually ventilated cages where many academic institutions keep experimental animals. Most of the inflammatory risk factors for increased severity of HI injury in human patients, such as maternal infection, will result in systemic inflammation in addition to CNS-specific recruitment of microglia. However, there is currently debate concerning the relevance of maternal inflammation to fetal brain damage. The debate intensifies when fetal systemic inflammation is contrasted with neuroinflammation. Although the majority of publications cited above administer pro-inflammatory agents by intracerebral injection, some studies have administered LPS by intravenous injection (reviewed in ). In fetal sheep, intravenous LPS causes white-matter specific damage, suggesting that systemic inflammation may be sufficient to trigger brain injury. However, the results of these experiments could be explained by a secondary neuroinflammatory response to systemic inflammation causing the white matter injury. Separating these parameters in future experiments will prove demanding.

31Data suggest that gene expression in mouse models of inflammation closely corresponds to that seen in humans, however, postnatal murine response to inflammation is likely different to that experienced by a human fetus. An important limitation of inflammatory studies is impact on cardiovascular function and the potential for secondary cerebral HI. Another is that varying results have been reported. This may be due to differences in LPS used between groups, purification method, or dose. Alternatively, there is increasing evidence that the considerable individual variability in response to clinical sepsis between patients is related to host genetics. Therefore, although inflammation is doubtless an important factor in neonatal brain injury, the variation between different models makes these methods difficult to evaluate.

32In many ways, non-rodent models are more representative of neonatal HI as it affects human patients. Here follows a brief flavor of techniques. Relatively few studies have investigated neonatal HI in primates. Classical studies asphyxiated term monkey fetuses by covering their heads with a rubber sac and clamping the umbilical cord. Fetuses resuscitated after 20 min had extremely high mortality. However, 12 min of asphyxia were required to produce any neuropathologic injury. The fetuses displayed damage predominantly within the brainstem. In a model of partial ischemia, pregnant females were rendered hypotensive during the third trimester. When blood oxygen saturation was reduced to 10% for 5 h, fetuses became profoundly acidemic. At birth, these displayed opisthotonus, decerebrate posturing, and convulsions. There is one model of white-matter injury based on baboons delivered prematurely by hysterotomy. Premature baboons were treated in an intensive care setting. Approximately half displayed white-matter injury. Analysis of behavior has not been undertaken.

33More work has been carried out in fetal sheep. Umbilical cord occlusion and term asphyxia has been carried out, repeated asphyxia at 5-h intervals resulted in injury to the striatum almost exclusively, whereas episodes of asphyxia repeated every 5 min caused diffuse and extensive damage to cortex, thalamus, and cerebellum in 40% of animals, and selective neuronal necrosis in the remainder. White matter injury has also been investigated, only those fetuses in which the mean arterial blood pressure fell below 30 mmHg exhibit brain damage, irrespective of hypoxia. Neuropathologic injury after carotid artery occlusion for 30 min caused both gray and white matter involvement, with cortical damage and selective neuronal necrosis in thalamus and striatum. Several laboratories have developed models in sheep after systemic endotoxemia. Unfortunately, no behavioral outcomes are currently available in sheep.

34Finally, other small laboratory animals have been investigated. Preterm rabbit fetuses exposed to sustained placental insufficiency via intrauterine occlusion of the descending aorta displayed significant alterations in motor responses to olfactory stimuli, coordination of suck and swallow, and marked hypertonia, reminiscent of spastic quadriplegia. Diffusion-weighted imaging detected a threshold in white matter loss below which all rabbit kits developed hypertonia,. Another promising model is that of the spiny mouse, a rodent which shows a similar level of brain development to a human neonate at birth, which also shows varied motor deficits and neuroanatomical pathology following neonatal HI.

35In brief, large animals can better replicate the conditions of a single human fetus exposed to a non-sterile environment. However, none of these models have access to the same extent of transgenic manipulation or validated behavioral tests that are possible in rodent studies. These models are invaluable for aiding comparison of brain development at birth which occur between species, whereas the relative immaturity of mouse and rat brains at birth introduce an unwelcome variable into these experiments. For the foreseeable future, insights from both rodent work and larger animals will be important for better understanding neonatal HI.

36One brain region exemplifying how much remains unknown about the developing brain’s response to HI, and the uncertainties involved in interpreting results from animal studies, is subplate. The subplate is an early-born transitory neuronal layer of cerebral cortex which serves an important role in the establishment of thalamocortical connectivity during development (reviewed in;;; ) (see Figure 4). Thalamocortical axons associate with and grow alongside subplate neurons in the developing cortex, and selective excitotoxic ablation of subplate disrupts thalamocortical connectivity in animal models.

37Subplate has been identified in a range of mammals by conserved molecular markers. Neuronal death occurs in infant subplate during normal development. However, the molecular mechanisms behind this programmed cell-death remain unknown. Functional roles for subplate in thalamocortical and corticothalamic development have been described, demonstrating lasting significance of this transient population.

38The susceptibility of subplate neurons to neonatal HI remains little understood. Early studies claimed subplate is selectively vulnerable. Cultured rat subplate neurons were more vulnerable to OGD compared to mixed cortical neurons. In rats exposed to systemic HI at E18, a significant decrease in Nurr1-expressing subplate neurons was documented by P2 and similarly in fetal sheep. However, Nurr1-expressing Layer VI neurons were also decreased. It is also possible that HI induced differential marker expression in subplate neurons, rather than cell death. In vitro electrophysiological recordings in neocortical slices from newborn rats have demonstrated a pronounced functional impairment of subplate neurons following OGD. Another influentiential study that claimed selective vulnerability of subplate to hypoxia used immunohistochemistry to detect cell-death in neurons labeled with BrdU at E10.5 following a modified Rice–Vannucci model in P1 rats. However, Layer VI neurons born at this date also expressed cell-death markers, suggesting subplate may not be selectively susceptible to HI injury. Conversely, a systematic quantification of cell death markers throughout subplate compared with other cortical layers was conducted using immunohistochemistry. Three subplate-specific marker positive populations showed little co-staining with caspase-3 in brains with mild to moderate HI lesions. In severely damaged cases, caspase-3 staining was found throughout the cortex. Therefore, layer-specific sensitivity of cortical neurons to HI remains debated. A double birthdating study, targeting subplate and layer V or VI combined with HI could resolve this issue. However, such an experiment has not yet been performed.

39Human literature supports the proposition that interstitial white matter, the equivalent of subplate in the postnatal human brain, shows structural damage as a result of neonatal injury. Tissue from preterm human infants with periventricular leukomalacia showed a deficit in the number of MAP-2 expressing neurons throughout the interstitial white matter. Scarring has been observed in interstitial white matter, alongside the expression of cell death markers, in brain tissue from human infants. The number of interstitial neurons present in white matter is known to peak at the developmental stage most sensitive to white matter injury. Immunohistochemical analysis on neonatal telencephalon samples obtained post-mortem from infants born at 25–32 weeks gestation showed a significant loss of GABA-ergic subplate neurons. Many reviews have highlighted the interstitial white matter as a site which should be more thoroughly investigated in neonatal brain injury. Several of these reviews propose a role for subplate in resultant cognitive and behavioral deficits, based on the established role of subplate in thalamocortical development. Unfortunately, post-mortem studies are unable to convey whether subplate pathology is responsible for any of the clinical outcomes in patients. The response of both human and rodent subplate to neonatal HI remains unclear, despite assertions of interest from the field. A systematic study of the subplate in vitro and in vivo following HI injury would provide valuable information. As subplate transiently integrates within the developing cortical circuitry (see Figure 5) interactions between subplate neurons and other cortical neurons, such as key interneuron populations, should also be further investigated.

40There are several reasons for believing that subplate could be critical in neonatal HI. One is the proposed transient secretory function. Subplate is enriched in CSPGs, whereas the adjacent intermediate zone is not. Chondroitin sulfate proteoglycans (CSPGs) are generally secreted from cells, and the subplate transcriptome catalogs a plethora of genes involved in production of extracellular matrix and proteoglycans. Additional genes with subplate-restricted expression in the cortex encode secreted proteins, including neuroserpin (Serpini1), neuronal pentraxin 1 (Nptx1), and insulin-like growth factor binding protein 5 (Igfbp5), several of which have been validated by immunohistochemistry. Connective tissue growth factor (CTGF), secreted extracellular matrix-associated protein involved in regulation of cellular adhesion, migration, mitogenesis, differentiation and survival, is also detectable in the subplate at E18, increasing in intensity at P3 and P8. It is unknown whether subplate-secreted proteins serve a protective function in the developing brain or not. Related to this secretory function, subplate neurons express relatively mature rER, an organelle essential for production of secreted proteins (reviewed in;;;; ). As a result of the cellular pressures of high protein production, cells activate a series of mechanisms referred to as the ER stress response. Nissl staining demonstrates enriched protein production in subplate at P8 in mouse, although staining is much fainter in adult. Morphology of subplate neurons is similar to that of rER-rich plasma cells under EM, and immunohistochemistry for ER stress marker binding immunoglobulin protein (BiP) confirmed that ER stress occurs in developing subplate. BiP protein synthesis is up-regulated in whole brain under stress conditions, such as glucose deprivation, hypoxia, or the presence of toxins. It is not known exactly which proteins are secreted throughout development by the subplate, but this enhanced metabolic stress during hypoxia and the potential for neuroprotective secretion reinforce the value of further study of subplate in neonatal HI models and human patients.

41The development of novel treatments to supplement the sole currently licensed therapy, hypothermia, is imperative. Since the discovery of this groundbreaking treatment, little progress has been made in identifying additive pharmacological therapies. Few potential treatments have translated to human clinical trials. Resuscitation at room temperature and xenon gas administration alongside hypothermia are the only therapies shown to have any additive effect. However, more recent publications have questioned the efficacy of xenon as an additive therapy alongside hypothermia. For example, randomized clinical trials have demonstrated that although xenon gas is a safe treatment, there is little or no therapeutic effect of combined hypothermia and xenon gas in moderate and severe cases of neonatal HI at 18 months follow-up. A similar experiment in rats found that xenon treatment made no difference to lesion size or neuronal cell numbers in cases of severe HI. Barbiturate anticonvulsants have no effect on long-term neurological development when given following neonatal HI (reviewed in;;;; ). Recent clinical studies suggest that high dose erythropoietin (EPo) treatment in term HI neonates reduces disability. However, even proponents of this potential treatment advise caution in interpreting these early results, and the therapeutic effect by no means completely prevents disability. Combination therapy of N-acetylcysteine, a free radical scavenger, and systemic hypothermia reduces infarct volume after focal HI injury. Another free radical scavenger, allopurinol, reduces cerebral edema and neuropathological damage. However, these treatments have only been trialed in animals, and the field is still awaiting a candidate neuroprotectice molecule which is both safe and effective in neonatal humans.

42Another therapeutic approach under development for neonatal HI is stem cell therapy (reviewed in;;;; ). These therapies make use of evidence that transplantation of human bone-marrow derived stem cells into the lesion can assist brain plasticity. Results of this therapy have been promising in animal models, however, the technique awaits validation in a clinical setting. Additionally, the use of stem cell therapy for cerebral palsy in human patients has produced mixed results.

43Therefore, new approaches are required to identify potential neuroprotective molecules as treatments for neonatal HI. Drawing on our detailed yet incomplete knowledge of the physiology of neonatal HI, several factors must be satisfied in a new potential therapy. All potential treatments should be safe for vulnerable neonates and not interfere with essential developmental milestones. This challenges the NMDA-inhibition strategies, as the glutamate system is essential for setting up normal synaptic plasticity within the developing brain. Treatments should also be specific, to avoid extreme adverse effects in these vulnerable infants. The ideal treatment would target molecules common to the excitotoxicity, oxidative stress and inflammation pathways. Targeting these common mediators would allow a single therapy to be efficacious against multiple mediators of brain damage, instead of merely eliciting a reshuffle of pathways to favor a different method of cell death. Here follows an outline of one potential therapy under development which may satisfy these theoretical restraints as a model to incite debate concerning how future therapies are identified. Enter: neuroserpin.

44Neuroserpin is a neuronally secreted serine-protease inhibitor enzyme with roles in cell death, neural plasticity, and microglial activation. Neuroserpin was first identified in the secreted product of cultured neuronal axons. Protein expression is primarily localized to neurons. The protein neuroserpin is encoded in mouse and human by the gene Serpini1. Serpini1 mRNA expression is specific to the CNS, both in mouse and human. In situ hybridization studies have demonstrated expression of Serpini1 throughout cortex, hippocampus, and olfactory bulbs, with scattered expression in cerebellum, pons, and thalamus from birth until adulthood. High Serpini1 expression has been identified in subplate relative to Layer VI of cortex in P8 mouse brain by microarray, confirmed by immunohistochemistry. Although Serpini1 expression has been demonstrated in human brain tissue, detailed maps of expression are currently missing. This highly specific localisation suggests that Serpini1/neuroserpin modulatory treatments may not create dangerous side effects in other vulnerable organs such as the cardiovascular and pulmonary systems.

45Neuroserpin is an inhibitory enzyme. Its primary target is the adult human stroke treatment, tissue plasminogen activator (tPA). Neuroserpin has also been shown to exhibit weak inhibition of plasmin and urokinase plasminogen activator. This function is conserved across species. Through inhibition of tPA, neuroserpin has the potential to influence many distinct molecular cascades. tPA, also secreted by neurons, has been shown to interact with a large number of pathways; activating NMDA receptors, activating microglia, and recruitment of cell death related cascades, in addition to its function of cleaving vascular thrombosis. The known components of the neuroserpin/tPA molecular cascade are summarized in Figure 6.

46Other, tPA-independent enzymatic pathways for neuroserpin have also been described. Cell lines over-expressing neuroserpin demonstrate an increase in N-cadherin protein expression and related cell adhesion, maintained when the tPA binding site of neuroserpin is mutated. In vitro, neuroserpin has been shown to prevent excitotoxic neuronal death induced by plasmin and kainic acid, and the 20 methionine residues present within neuroserpin have been claimed to convey protection against oxidative stress, and exogenous neuroserpin has been shown to possess anti-inflammatory properties. The molecular cascades behind these tPA independent functions are not well understood. Therefore, neuroserpin has the pedigree to target multiple cell death pathways occurring in the brain during neonatal HI.

47Neuroserpin’s physiological actions can be broadly categorized into three major pathways: cell death, neuronal plasticity, and immune cell activation. These closely follow the major classes of molecular change proposed by pathological theories of neonatal HI, suggesting that neuroserpin offers the potential to target multiple brain-damage pathways.

48Neuroserpin has been implicated in protection against neuronal death. Exogenous neuroserpin administration in vitro decreases apoptosis caused by tPA, NMDA, kainic acid, and OGD. In addition, aberrant initiation of neuroserpin expression in cancer cells preserves the tumor and has been linked with increased treatment-resistance in prostate cancer. Exogenous neuroserpin reduces spread of kainate-induced seizures in mouse, and decreases the expression of cell death markers.

49Of the three classes of neuroserpin function, its role in inflammation is the least well understood. When applied to vascular plaques in vitro, recombinant neuroserpin reduced T-Cell lymphocyte invasion. Administration of intracerebral neuroserpin directly following adult HI stroke in mouse, showed a qualitative decrease in the volume of infarct infiltrated by activated microglia, and lower microglial inflammatory marker expression. In human adult stroke patients, higher levels of neuroserpin in blood samples correlate with lower levels of immune marker proteins. There is considerable debate whether microglial activation is beneficial or detrimental in neonatal HI, so understanding the role of neuroserpin in balancing this cascade could provide important data. The lack of a complete molecular network explaining these anti-inflammatory effects highlights the complexity of the pathways involved in cell death and inflammation. Understanding the underlying explanation for these changes will be an important preliminary to administration in human infants who do not live in the highly sterile environments maintained for experimental animals.

50In addition, neuroserpin is involved in neuronal plasticity. This could be problematic in the development of neuroserpin as a neonatal HI treatment for human patients, as the neonatal human brain circuitry is undergoing a great deal of developmental plasticity modifications (reviewed in;; ). The first evidence that neuroserpin may have a role in neuronal plasticity originated from studies of endocrine cell lines, which grow neurite-like processes when neuroserpin is administered to their media. At the molecular level, neuroserpin secretion from dense core vesicles is enhanced by depolarisation in cultured neurons. Monocular enucleation at P11 in mouse led to decreased Serpini1 mRNA expression in contralateral primary visual cortex compared to the control hemisphere. The relative importance of this molecular cascade for human plasticity is as yet unclear.

51Neuroserpin has been studied extensively in cell culture models of HI stroke. Administration of neuroserpin protects mouse cortical neurons in vitro against NMDA-induced excitotoxicity, but not AMPA-induced excitotoxicity. Further, neuroserpin is protective in an in vitro model of HI, OGD. Both neurons and astrocytes cultured in OGD undergo less apoptosis and do not exhibit damaged neural processes when exogenous neuroserpin is administered following OGD. Hippocampal neurons from both wild-type and tPA knockout mice were protected from OGD, plasmin-mediated cell death and kainic acid by neuroserpin administration, demonstrating that neuroserpin’s neuroprotective role is not exclusively dependent on tPA inhibition.

52Neuroserpin is also neuroprotective in vivo. The major model used to simulate hypoxic-ischaemic stroke in adult rodents is analogous to the Rice–Vannucci model described above for neonates. Adult rats injected with neuroserpin before left common carotid artery ligation followed by acute hypoxia showed a decrease in infarct volume compared to controls, in addition to a decrease in expression of apoptosis markers. Neuroserpin knockout mice show increased lesion volumes following adult HI, whereas transgenic mice expressing six-times normal neuroserpin expression demonstrate smaller infarct volumes. Exogenous neuroserpin injection is also protective against NMDA injection in live adult rat. Currently, the only evidence for this has come from adults models of HI stroke, far from conclusive evidence of neonatal protection due to the differences in neuroanatomy and neurochemistry between the developing and adult brain. However, this work remains of utility, as at the very least investigating the neuroserpin-tPA system in neonatal models will add to understanding of the differences between neonatal and adult response to HI.

53Neuroserpin administration in rats increases the window for effective tPA treatment in a model of adult stroke. Increased tPA expression is found in neuroserpin knockout mice undergoing adult HI injury compared to controls. However, exogenous neuroserpin is neuroprotective in tPA knockout mice. Ischaemic-reperfusion-induced injury in the retina of tPA knockout mice demonstrated rescued retinal apoptotic marker expression when injected with neuroserpin. Therefore, the neuroprotective properties of neuroserpin in adult HI appear to involve both neuroserpin’s tPA-dependent and tPA-independent molecular cascades.

54A role for neuroserpin in human adult HI stroke has also been described. Correlative studies have shown that high concentrations of neuroserpin in blood samples from human patients are associated with better functional outcome and reduced inflammation. However, attempts to find a relationship between neuroserpin polymorphisms in humans and likelihood of stroke have produced little evidence of protective neuroserpin variants. Mutations in the neuroserpin gene have been associated with two rare hereditary disorders; individuals present either with epilepsy or dementia with neuroserpin inclusion bodies. It is not currently known whether these disorders are caused by novel functions of the mutated protein or by loss of physiological functions.

55The evidence above from adult models of HI stroke is far from conclusive evidence of neonatal protection due to differences between the developing and adult brain. However, this work remains of utility, as at the very least investigating neuroserpin in neonatal models will add to understanding of the differences between neonatal and adult response to HI.

56Evidence for a role of neuroserpin in neonatal HI is not entirely absent. A patent application published online claims that neuroserpin injection 4 h after neonatal HI in rat pups reduced infarct volume, but had no effect on LPS-sensitized HI. However, the small number of published rodent microarrays following neonatal HI have failed to detect significant changes in Serpini1 expression. This could be explained by the delay between HI injury and sample collection, 6 h to 7 days post injury in this sample, which may be too early or too late to expect to see genomic changes in Serpini1 expression. Also, microarray studies cannot reveal whether neonatal HI causes differences in neuroserpin translation or secretion, neither of which have yet been studied in neonatal HI.

57Despite the lack of evidence for neuroserpin’s direct role in neonatal HI, substantially more has been published documenting effects of its primary molecular target, tPA, in neonatal HI. tPA is broadly considered to be deleterious to cerebral recovery from neonatal HI. In P7 rats which underwent unilateral common carotid artery ligation, tPA treatment was found to impair blood flow throughout the brain, an effect which was rescued by injection of the tPA inhibitor α2-antiplasmin. Release of tPA by the cerebral microvasculature, as seen following neonatal HI, has also been shown to compromise the glucose content of the extracellular medium in neonatal mouse cortical culture. Administration of tPA inhibitor Plasminogen-activator-inhibitor 1 (PAI1) reduces locomotor disorder and white matter damage in LPS-sensitized neonatal HI rats. This pathway is relatively understudied in neonatal HI research, despite the potential to target a multitude of injury-relevant molecules. Drawing attention to molecules like neuroserpin and tPA, potential common moderators of injury pathways, will be essential to the development of new efficacious pharmacological treatments.

58Neonatal HI remains the most common cause of infant death and disability globally. Reducing the burden of morbidity should be a high priority for biomedical research. A dearth of treatments are currently available postnatally for these vulnerable infants, restricted to only hypothermia, a breakthrough treatment which is not fully effective in all cases, with no currently licensed additive pharmacological treatments. A complex network of interacting molecular cascades, including excitotoxicity, oxidative stress, and inflammation contribute to the gradually developing pattern of neuronal cell death seen in asphyxiated neonates. Targeting common mediators of these pathways with specific targets linked to far-reaching effects offers a potential approach to generating new pharmacological treatments to exact neuroprotection in these neonatal patients.

59All authors listed have made substantial, direct, and intellectual contribution to this work, and approved its final version for publication. LM, LS, AH-S, and ZM: conceived the review focus, conducted literature review, evaluated the literature. LM: reviewed literature, wrote first draft, critically revised the first draft, and finalized the manuscript. LS, AH-S, and ZM: gave feedback, and finalized the manuscript. All authors approved final version of manuscript.

60The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.