Clinical Syndromes

Neonatal encephalopathy and seizures are the most overt manifestation of the severity of brain injury. Clinical signs and symptoms depend on the severity, timing, and duration of the insult, as well as the newborn’s maturity, even within ages considered “full term.” It is equally important to note that not all newborns with significant acquired brain injuries present with an easily recognizable encephalopathy or seizures. Some conditions, such as stroke and perinatal white matter injury, discussed in Chapters 18 and 19, may only have subtle manifestations that may not be recognized on clinical examination. This chapter will focus on the clinical syndrome of neonatal encephalopathy secondary to hypoxia-ischemia. The diagnosis and management of neonatal seizures is discussed in Chapter 16.

Neonatal Encephalopathy

The major components of the syndrome of “neonatal encephalopathy” include: alertness, tone, respiratory status, reflexes, feeding, and seizures (Table 17-1). The severity of brain injury, reflecting the duration and magnitude of the hypoxic-ischemic insult, determines the severity of evolution of this clinical encephalopathy. In newborns with moderate to severe encephalopathy, symptoms usually evolve over days, making serial detailed examinations important. As with any neurologically ill newborn, the baby’s gestational age must be considered in the interpretation of physical findings. As described by Volpe, there is a characteristic progression of signs in newborns with a severe hypoxic-ischemic insult [Volpe, 2008]. The affected neonate exhibits a depressed level of consciousness from the very first hours of life. Periodic breathing with apnea and bradycardia often heralds this initial presentation. Hypotonia with decreased movement is almost universally found. In the first day of life, the pupillary response is often preserved and abnormalities of eye movements are not detected. In severely injured newborns, seizures may be seen within 6–12 hours after birth. Seizures at this stage are often subtle, manifesting as ocular movements, lip smacking, apnea, or bicycling movements of the extremities. The nursing staff should be alerted to identify and document paroxysmal behaviors. Multifocal or focal clonic seizures may also occur, and often indicate focal cerebral infarction. In the latter hours of the first day of life, there may be a transient increase in the level of alertness that is not accompanied by other signs of neurological improvement. This should not be falsely reassuring, as this apparent increase in alertness is frequently accompanied by more seizures and apnea, a shrill cry, and jitteriness. With careful bedside examination, weakness in the proximal limbs and increased muscle stretch reflexes may be observed, but in the very severely injured newborn, diffuse weakness with absent movements and reflexes is common.

By the third day of life in newborns with severe brain injury, the level of consciousness deteriorates with symptoms of respiratory failure and other signs of brainstem dysfunction. During this period, cerebral edema resulting from hypoxia-ischemia is maximal and can further impair cerebral blood flow secondary to increased intracranial pressure. Most clinicians avoid treatment of cerebral edema in this setting, as many interventions such as corticosteroids, hyperventilation (PaCO₂ 20–25 mmHg), furosemide, or mannitol, may be harmful and no studies have shown proven benefit. Those newborns surviving beyond the third day of life begin to show improved alertness. Yet hypotonia and weakness in proximal limbs, face, and bulbar musculature persist. While less intensive care may be required with this improved level of consciousness, the clinician must be vigilant for feeding difficulties and the risk of aspiration as the newborn’s suck and swallow responses often remain impaired.

Documenting this clinical evolution is a critical component of the evaluation, and use of a simple encephalopathy score as a bedside tool can help the clinician standardize assessment of encephalopathic newborns, monitor the evolution of the clinical syndromes, and help in systematically identifying neonates who require therapeutic intervention (see Table 17-1) [Miller et al., 2004].

Table 17-1 The Encephalopathy Score

(From Miller et al. Clinical signs predict 30-month neurodevelopmental outcome after neonatal encephalopathy. Am J Obstet Gynecol 2004;190(1):93–99.)

Subtle Neonatal Syndromes

Stroke and white matter injury in the term newborn may present with clinical signs that are so subtle that they remain undetected for months or years. Advances in brain monitoring (amplitude integrated EEG, prolonged video-EEG monitoring) and brain imaging (MRI) in the neonatal intensive care unit have increased recognition of these injuries. However, both stroke and white matter injury may present in the neonatal period with an overt encephalopathy [Li et al., 2009; Ramaswamy et al., 2004]. These conditions share many of the risk factors of “global” hypoxic-ischemic brain injury considered in this chapter.

Clinical Management

As many etiologies of neonatal encephalopathy have specific therapies, the clinician’s initial task is to determine the underlying etiology through careful history taking, neurological examination, and laboratory and brain imaging studies. The history should elicit indicators of intrauterine distress that may have contributed to decreased placental or fetal blood flow: fetal heart tracing abnormalities, passage of meconium, or a history of a difficulty in labor or delivery. The Apgar score will indicate who is at risk for mortality. The details of the delivery-room resuscitation, medications, and ventilatory support should be noted. Because the clinical signs of encephalopathy and seizures are often not specific for an etiology, laboratory tests are critical to exclude reversible causes of neonatal encephalopathy. The management of moderate or severe encephalopathy should occur in a neonatal intensive care unit in close collaboration with a neonatologist. Immediate management requires securing an appropriate airway and maintaining adequate circulation. Ventilatory support with mechanical ventilation or continuous positive airway pressure (CPAP) is often required. Metabolic complications, such as hypoglycemia, hypocalcemia, hyponatremia, and acidosis, frequently accompany hypoxic-ischemic encephalopathy (HIE), and should be identified and treated. Liver enzymes and serum creatinine levels should be performed to detect injury to other organs, while serum ammonia and lactate levels can screen for possible inborn errors of metabolism. Lumbar puncture to evaluate for intracranial infections should be performed if the history is not typical for HIE, or if a clinical suspicion of infection exists. If infection is suspected, ampicillin and gentamicin are started in addition to acyclovir if herpes simplex virus infection is a consideration. If the history, examination, or initial laboratory investigation points to an inborn error of metabolism, early treatment is crucial and a biochemical geneticist should be consulted. Additional diagnostic tests, such as serum amino acids and urine organic acids, as well as specific management strategies, are required (detailed in Chapter 20). The diagnosis of a severe intracranial hemorrhage should prompt consultation with a neurosurgeon to manage raised intracranial pressure from mass effect or hydrocephalus; platelet levels and coagulation function should be measured. Since the clinical syndrome evolves considerably over the first 72 hours of life, management of specific complications, such as respiratory compromise or seizures, can often be anticipated.

Investigation of term newborns with encephalopathy addresses three primary questions:

Addressing these questions is critical for the application of “neuroprotection” strategies, such as hypothermia [Eicher et al., 2005; Gluckman et al., 2005; Shankaran et al., 2005; Azzopardi et al., 2009]. To assess the encephalopathic term newborn, diagnostic tools available to the clinician include clinical features and biochemical and electrophysiological tests. However, the severity of brain injury in term asphyxiated newborns is not reliably predicted by clinical indicators commonly used during the first days of life, such as umbilical cord pH and Apgar scores [Shevell et al., 1999]. On the other hand, the severity of clinical encephalopathy is a strong predictor of neurodevelopmental outcome [Miller, 2004]. However, while the risk of motor and cognitive deficits appears to be minimal in mild encephalopathy, and pronounced at the severe end of the spectrum, it is inconsistent in neonates with moderate encephalopathy [Sarnat and Sarnat, 1976; Robertson et al., 1989; Dixon et al., 2002; Marlow et al., 2005]. Newborns with moderate encephalopathy are thus the most likely to benefit from the improved prognostic capabilities of brain imaging.

Brain Imaging of Newborns with Encephalopathy

Current guidelines for neuroimaging term newborns with encephalopathy suggest a noncontrast computed tomography (CT) scan to detect hemorrhage in infants with a history of significant birth trauma, and evidence of low hematocrit or coagulopathy [Ment et al., 2002]. If CT findings cannot explain the newborn’s clinical status, then MRI is recommended. For other encephalopathic term newborns, MRI is recommended between days 2 and 8 to assess the location and extent of injury [Ment et al., 2002]. In a recent study, the neuroimaging findings from a group of 48 term newborns with encephalopathy uniformly scanned with CT and MRI (T1 and T2-weighted images) with diffusion-weighted imaging (DWI) on the third day of life were compared [Chau et al., 2009]. On the third day of life, both CT and MRI with DWI reliably identify injury to the basal nuclei. However, DWI more readily detects cortical injury and focal and multifocal lesions, such as strokes and white matter injury, than either CT or conventional MRI. The choice of the best neuroimaging technique must balance the risk of transport and sedation involved in MRI against the risk of ionizing radiation with CT [Brenner et al., 2003; Berrington de Gonzalez and Darby, 2004; Hall et al., 2004]. With the development of MR-compatible incubators and monitoring equipment [Dumoulin et al., 2002; Bluml et al., 2004], as well as improved capacity to scan newborns without pharmacological sedation, MRI should now be the modality of choice when possible. MRI with DWI appears to be the most sensitive imaging study to detect abnormalities associated with other causes of neonatal encephalopathy, such as cerebral dysgenesis, infections, stroke, and metabolic disorders [Volpe, 2008; Cowan et al., 2003]. In order to confirm the diagnosis of hypoxic-ischemic brain injury and determine the extent of injury, MRI and DWI are optimally obtained between 3 and 5 days of life in term newborns with encephalopathy [Chau et al., 2009; Barkovich, 1997; Rutherford et al., 2004; Barkovich et al., 2006]. In newborns treated with hypothermia, further studies are needed to determine the optimal timing of MRI. The importance of rigorous imaging protocols with appropriate quality controls, and high quality neuroradiological review, must be emphasised.

Motor Function

In term survivors of hypoxic-ischemic brain injury, the risk of cerebral palsy or severe disability may involve more than one-third of affected newborns, and is most common in those with a severe encephalopathy [Volpe, 2008; Dixon et al., 2002; Barnett et al., 2002]. Spastic quadriparesis is the most common type of cerebral palsy, although athetoid or spastic hemiparesis also occurs. The diagnosis and management of cerebral palsy are addressed in Chapter 69. A complete assessment of neurosensory and cognitive functions is critical in children with cerebral palsy [Marlow et al., 2005]. Minor motor impairments that do not meet diagnostic criteria for cerebral palsy [Shevell et al., 1999] are diagnosed in more than one-third of children with moderate encephalopathy, and in more than one-quarter of children with mild encephalopathy [Van Kooij et al., 2008].

Vision and Hearing

Severe visual impairment occurs in up to one-quarter of children after moderate or severe encephalopathy [Robertson and Finer, 1985; Shankaran et al., 1991]. This may be due to injury to the posterior visual pathway, including the primary visual cortex, resulting in “cortical visual impairment” [Van Hof-van Duin and Mohn, 1984]. Injuries to the basal nuclei may also affect acuity, visual fields, or stereopsis (depth perception) [Mercuri et al., 1997]. Sensorineural hearing loss, likely secondary to brainstem injury, is also seen following neonatal encephalopathy [Robertson and Finer, 1985; Robertson et al., 1989], affecting 18 percent of survivors of moderate encephalopathy without cerebral palsy [Lindstrom et al., 2006].

Cognition

Overall, cognitive deficits are seen in 30–50 percent of childhood survivors of moderate neonatal encephalopathy [Dilenge et al., 2001]. Intellectual performance in children with severe encephalopathy without cerebral palsy is also affected [Marlow et al., 2005]. School-age survivors of moderate neonatal encephalopathy are more likely to have difficulties with reading, spelling, and arithmetic, or require additional school resources [Moster et al., 2002; Robertson et al., 1989]. Cognitive deficits, such as those in language and memory, may be seen, even when IQ scores are “normal.” [Marlow et al., 2005]. Behavioral difficulties, such as hyperactivity and emotional problems, should also be considered, even in individuals without motor disability [Marlow et al., 2005].

Brain Imaging and Outcome

The pattern of brain injury on neuroimaging conveys important prognostic information regarding the “pattern” of neurodevelopmental abnormalities. The basal nuclei pattern of injury and abnormal signal intensity in the posterior limb of the internal capsule are both predictive of severely impaired motor and cognitive outcomes [Rutherford et al., 1998; Miller et al., 2005]. The cognitive deficits associated with this pattern are not surprising, given the common involvement of the cerebral cortex [Miller et al., 2005] and cerebellum [Le Strange et al., 2004; Sargent et al., 2004]. In contrast, the watershed pattern is associated with cognitive impairments that are not necessarily accompanied by major motor deficits [Miller et al., 2005]. Importantly, the cognitive deficits following the watershed pattern may only be evident after 2 years of age [Miller et al., 2005]. In survivors of neonatal encephalopathy without functional motor deficits assessed at 4 years of age, the severity of watershed-distribution injury was most strongly associated with impaired language skills [Steinman et al., 2009]. While neurodevelopmental outcomes may be significantly better than anticipated, neurological deficits may also be found in some newborns whose brain imaging studies are normal [Bax et al., 2006]. More subtle brain injuries associated with later neurodevelopmental deficits, such as white matter injuries or hippocampal volume loss, may only be detectable with quantitative brain imaging techniques [Miller et al., 2002a; Nagy et al., 2005; Gadian et al., 2000].

Cerebral Blood Flow and Energy Metabolism

Disruption of cerebrovascular autoregulation has been implicated as an important factor in the pathophysiology of neonatal hypoxic-ischemic brain injury. It is widely accepted that preterm infants have a “pressure-passive” cerebral circulation; however, term infants may remain at risk for impairment of cerebrovascular autoregulation [Boylan et al., 2000] and susceptibility to cerebral ischemia with fluctuations in systemic blood pressure. Several basic physiologic mechanisms may contribute to impaired autoregulation in the neonate. Increased expression of inducible and neuronal isoforms of nitric oxide synthase (iNOS and nNOS), as well as endothelial NOS, may narrow the autoregulatory window, and downregulation of prostaglandin receptors in response to high circulating prostaglandin levels may blunt the prostaglandin-mediated vasoconstrictive response to hypertension and thereby contribute to inappropriately increased cerebral blood flow [Chemtob et al., 1996]. After an ischemic insult, the neonate remains at high risk for further damage in the acute recovery phase because neonatal encephalopathy is often associated with blood pressure fluctuations.

An inadequate supply of glucose or alternate substrates plays a pivotal role in hypoxic-ischemic neuronal cell death. Although overall metabolic demands are lower in the neonatal than in the adult brain, during periods of rapid brain growth, particularly the perinatal period, metabolic needs rise. The pattern of injury after hypoxia-ischemia can be explained in part on the basis of this metabolic demand; brain regions most susceptible to hypoxic-ischemic injury in the term infant (e.g., subcortical gray matter structures such as the basal ganglia and thalamus) are the same regions that are most vulnerable to mitochondrial toxins. Brain development is associated with a transition from the ability to use glucose and ketones as energy substrates in the neonate to an absolute requirement for glucose in the adult. The immature brain can use lactate as an alternate fuel source to some degree, and the deleterious effects of lactate accumulation after hypoxia-ischemia therefore may be attenuated in the neonate compared with the adult. However, normal maturation is characterized by limitations in glucose transport capacity and increased use of these alternative fuels such as lactate. The inability to transport glucose across the blood–brain barrier threatens cerebral glucose utilization. These factors illustrate the importance of understanding the use of glucose, lactate, and ketones in the newborn brain under normal and pathologic conditions [Vannucci and Vannucci, 2000; Vannucci and Hagberg, 2004].

Excitotoxicity

Glutamate can activate a variety of excitatory amino acid receptors that are broadly classified based on their selective responses to specific agonists (e.g., NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid [AMPA], kainate) or their signaling mechanisms (i.e., ionotropic [ligand-gated ion channel] or metabotropic [G-protein-coupled]). Excitatory amino acid neurotransmission plays a pivotal role in brain development and in learning and memory. A substantial body of data has emerged over the past 30 years documenting the fact that overactivation of excitatory amino acid receptors (i.e., excitotoxicity) contributes to neurodegeneration in a broad range of acute and chronic neurologic disorders [Johnston, 2005].

Most information about mechanisms of excitotoxicity comes from experimental studies, but studies in the human neonate have provided some evidence to support the hypothesis that hypoxic-ischemic brain injury disrupts brain glutamate metabolism. Elevated levels of cerebrospinal fluid glutamate have been documented (by direct cerebrospinal fluid measurements and by proton MRS) in infants with severe hypoxic-ischemic injury [Pu et al., 2000]; cerebrospinal fluid levels of excitatory amino acids are directly proportional to the severity of clinical encephalopathy [Hagberg et al., 1997; Riikonen et al., 1992].

Two closely linked mechanisms contribute to ischemia-induced increases in synaptic glutamate: increased efflux from presynaptic nerve terminals and impaired reuptake by glia and neurons. The initial increase in efflux is mediated by a calcium-dependent process through activation of voltage- dependent calcium channels; later, calcium-independent efflux is thought to be mediated primarily by functional reversal of glutamate transporters. Removal of glutamate from the synaptic cleft depends primarily on energy-dependent glutamate transporters, which are predominantly glial. Any pathophysiologic process that depletes energy supply (e.g., hypoxia-ischemia, hypoglycemia, prolonged seizures) will disrupt these mechanisms and result in increased synaptic glutamate accumulation [Johnston, 2005].

The structure and function of the NMDA receptor channel complex are developmentally regulated. The receptor possesses multiple functional sites that recognize glutamate, co-agonists, modulatory molecules such as glycine, dissociative anesthetics, redox agents, steroids, zinc, magnesium (which blocks permeability to calcium), and a cation-selective ion channel that admits Na⁺, K⁺, and Ca²⁺. When the neurotransmitter recognition site is activated by glutamate, the ion channel allows influx of calcium and sodium. The resulting increase in intracellular calcium is the stimulus for a multitude of downstream events, including transcription factor modulation, cell cycle regulation, and DNA replication. The NMDA receptor is relatively overexpressed in the developing brain compared with the adult brain; in postnatal day 6–14 rats (which approximates to the term human neonate), the NMDA receptor is expressed at 150–200 percent of adult levels. In humans, receptor expression is significantly higher at term than in the adult [Johnston, 2005]. The particular combination of subunits determines the NMDA receptor’s functional state, and the predominating combination in the perinatal period seems to favor a more prolonged and pronounced calcium influx. In the setting of hypoxia-ischemia, NMDA receptor overactivation leads to massive Na⁺ and water influx, cell swelling, elevated intracellular calcium and its associated mitochondrial dysfunction, increased nitric oxide production, increased phospholipid turnover and accumulation of potentially toxic free fatty acids, and cell death by apoptotic or necrotic mechanisms. However, ischemia and energy failure also result in cation influx by non-NMDA-mediated mechanisms.

In experimental rodent models, there is compelling evidence that susceptibility to NMDA- and AMPA-mediated excitotoxicity peaks in the immature and that treatment with NMDA and AMPA receptor antagonists confers robust protection against neonatal hypoxic-ischemic brain injury [Johnston et al., 2001]. However, concerns have been raised that NMDA receptor antagonists might have specific risks in the immature brain; blockade of NMDA synaptic activity could disrupt critical neurodevelopmental processes. Whether AMPA antagonists, which can block neuronal and white matter hypoxic-ischemic damage in neonatal rodent models, have fewer potential risks in the immature brain than do NMDA antagonists is an important question for study [Silverstein and Jensen, 2007].

There has been considerable interest in evaluating the neuroprotective efficacy of magnesium sulfate in experimental and clinical models of neonatal brain injury. This interest stems from the intrinsic potentially neuroprotective properties of Mg²⁺ (including blockade of NMDA receptor activation), and the many years of clinical experience in safe use of magnesium sulfate in obstetric practice. Experimental studies provide evidence that pretreatment with magnesium sulfate can limit subsequent neonatal hypoxic-ischemic injury, but treatment after hypoxia-ischemia is of limited benefit [Greenwood et al., 2000; Turkyilmaz et al., 2002]. Magnesium sulfate has many effects, including blockade of NMDA receptors. It has been found give protection in some animal models of white matter damage [Turkyilmaz et al., 2002; Marret et al., 1995; Spandou et al., 2007], but did not affect cerebrospinal fluid levels of excitatory neurotransmitters in asphyxiated human neonates [Khashaba et al., 2006]. In addition, in a multicenter clinical trial of mothers treated with magnesium who were at risk for preterm delivery, no perinatal side effects were seen and there was some benefit in the neurodevelopment of survivors [Crowther et al., 2003]. A recent study, the BEAM trial, showed that exposure to magnesium sulfate before anticipated early preterm delivery did not reduce the combined risk of moderate or severe cerebral palsy or death, although the rate of cerebral palsy was reduced among survivors [Rouse et al., 2008].

Oxidative Stress

Oxidative stress describes the alterations in cellular milieu that result from an increase in free radical production as a result of oxidative metabolism under pathologic conditions. In the brain injured by hypoxia-ischemia, excitotoxicity and oxidative stress are inextricably linked. In cells with normally functioning mitochondria, more than 80 percent of available oxygen is reduced to energy equivalents (ATP) by cytochrome oxidase. The rest is converted to superoxide anions, which, under physiologic conditions, are reduced to water by enzymatic and nonenzymatic antioxidant mechanisms. An inevitable consequence of mitochondrial dysfunction is an accumulation of superoxide, and any process that depletes antioxidant defenses will result in the default conversion of superoxide to even more reactive species, such as the hydroxyl radical [Ferriero, 2001]. The concept of ischemia-reperfusion injury (i.e., progression of tissue injury with reoxygenation after ischemia) [Inder and Volpe, 2000] is fundamental to the understanding of oxidative stress. This mechanism is not limited to the brain, but excitotoxic mechanisms in the brain can amplify these processes. Excitotoxicity causes energy depletion, mitochondrial dysfunction, and cytosolic calcium accumulation, leading to the generation of free radicals, such as superoxide, nitric oxide derivatives, and the highly reactive hydroxyl radical.

With reoxygenation, mitochondrial oxidative phosphorylation is overwhelmed and reactive oxygen species accumulate. Intrinsic antioxidant defenses are depleted, and free radicals directly damage multiple cellular constituents (lipids, DNA, protein) and can activate pro-apoptotic pathways. The brain is particularly susceptible to free radical attack and lipid peroxidation, and vulnerability is magnified in the immature brain. Contributing factors include a high polyunsaturated fatty acid content, high level of lipid peroxidation (particularly in response to hypoxic stress), immaturity of antioxidant defense enzymes, and high free iron concentrations, compared with the adult brain [Ferriero, 2001]. The level of free iron, which catalyzes the production of various reactive oxygen species, is increased in the plasma and cerebrospinal fluid of asphyxiated newborns [Ogihara et al., 2003]. The deleterious effects of abundant iron and immaturity of the enzymatic oxidant defenses of the immature brain are tightly interrelated. Free radical scavengers (e.g., alpha-phenyl-n-tert-butyl-nitrone [PBN], a spin-trap agent that converts free radicals to stable adducts) and metal chelators (e.g., deferoxamine) can protect neurons from injury mediated by hydrogen peroxide in vitro and in vivo. These agents also protect neurons from NMDA-induced toxicity, providing complementary evidence of the pathophysiologic link between excitotoxicity and oxidative stress [Peeters-Scholte et al., 2003].

Nitric oxide metabolism provides another critical link between excitotoxicity and oxidative injury in the hypoxic-ischemic injured brain. Nitric oxide is produced constitutively in endothelium, astrocytes, and neurons in response to an increase in intracellular calcium. Hypoxic-ischemic increases in nitric oxide production have multiple potential beneficial and detrimental effects. Nitric oxide regulates vascular tone, influences inflammatory responses to injury, and directly modulates NMDA receptor function [Sorrentino et al., 2004]. In neonatal rodent brain, striatal neurons that express nNOS are selectively resistant to hypoxia-ischemia injury, providing a source for NMDA receptor-mediated regulation of nitric oxide production. Disruption of the nNOS gene and pharmacologic inhibition of nNOS (reviewed in Gonzalez and Ferriero) ameliorate neonatal hypoxia-ischemia injury [Gonzalez and Ferriero, 2008]. Other nitric oxide synthase isoforms contribute to injury via inflammation (iNOS) and may potentially provide another crosstalk for protection therapies. Early endothelial NO is protective by maintaining blood flow, but early neuronal NO and late inducible NO are neurotoxic by promoting cell death [Iadecola et al., 1997]. Brain iNOS is induced in multiple cell types during upregulation of the pro-inflammatory pathway after brain injury [Higuchi et al., 1998], enhancing excitotoxicity by modifying binding to NMDA receptors [Ishida et al., 2001].

Neuroprotection by selective inhibition of nNOS or iNOS has been demonstrated [van den Tweel et al., 2005]. Regions expressing nNOS correspond to those that express NMDA receptors and correlate with regions of neurotoxicity both in vivo and in vitro [Black et al., 1995; Ferriero et al., 1996; Dawson et al., 1993]. Destruction of neurons containing nNOS with local injections of quisqualate prior to hypoxia-ischemia results in lower injury scores and less infarction [Ferriero et al., 1995]. Likewise, neonatal mice with targeted disruption of the nNOS gene, when subjected to a similar hypoxic-ischemic insult, are markedly protected from injury [Ferriero et al., 1996], but nonspecific blockade of both nNOS and eNOS (endothelial NOS) in sheep with NG-nitro-L-arginine (L-NNA) was not protective [Marks et al., 1996]. Few studies have been performed in asphyxiated human newborns in relation to cerebral NO production. Cerebrospinal fluid NO levels increase with severity of HIE at 24–72 hours after asphyxia [Ergenekon et al., 1999], with increased NO and nitrotyrosine levels in spinal cord as well [Groenendaal et al., 2008]. Initial results in premature infants treated with inhaled NO for prevention of bronchopulmonary dysplasia showed reductions in ultrasound-diagnosed brain injury and improvements in neurodevelopmental outcomes at 2 years of age, but long-term results are pending [Schreiber et al., 2003; Ballard et al., 2006].

Several other antioxidant strategies that either block free radical production or increase antioxidant defenses have been studied. Melatonin is an indoleamine that is formed in higher quantities in adults and is a direct scavenger of reactive oxygen species and NO. It has been found to provide long-lasting neuroprotection in experimental hypoxia-ischemia and focal cerebral ischemic injury [Carloni et al., 2008; Koh, 2008]. Human neonates treated with melatonin were also found to have decreased proinflammatory cytokines [Gitto et al., 2004, 2005]. Allopurinol has mixed effects which have shown promise in animal and human studies. Xanthine oxidase-derived superoxide and H₂O₂ react with NO to form damaging RNS (reactive nitrogen species). Allopurinol functions by reducing free radical production via xanthine oxidase inhibition, while also scavenging hydroxyl radicals. Short-term benefits have also been seen in neonates undergoing cardiac surgery for hypoplastic left heart syndrome [Clancy et al., 2001]. Early allopurinol in asphyxiated infants improved short-term neurodevelopmental outcomes and decreased serum NO levels after administration; however, no improvement in long-term outcomes was seen for later treatment after birth asphyxia, perhaps because only a very brief window for benefit exists [Benders et al., 2006].

Inflammation

Nelson and colleagues first reported a link between elevated neonatal blood levels of a broad range of serum proinflammatory cytokines and subsequent diagnosis of spastic diplegia [Nelson et al., 1998]. Cytokines are polypeptides that act systemically and locally to mediate and regulate multiple components of inflammation. Cytokines that have been strongly implicated as mediators of brain inflammation in neonates include interleukin (IL)-1β, tumor necrosis factor (TNF)α, IL-6, and membrane co-factor protein-1 [Foster-Barber and Ferriero, 2002]. After an asphyxial episode, there are many potential sources of plasma cytokines, including injured endothelium and acutely injured organs, such as the brain by means of a disrupted blood–brain barrier. Measurements of cerebrospinal fluid and plasma levels of several cytokines in asphyxiated term infants suggest that the injured brain can be the source of acutely elevated cytokine levels [Silveira and Procianoy, 2003]. A more recent study links IL-6 with cerebral palsy, giving credence to the fact that there is an underlying genetic susceptibility in at-risk newborns [Wu et al., 2009].

The observed relation between maternal infection and neonatal brain injury could be explained by a number of mechanisms. Experimental studies provide support for the hypothesis that a proinflammatory milieu increases the susceptibility of the neonate to hypoxic-ischemic brain injury. In fetal or neonatal models, maternal infection is commonly simulated by injecting the potent proinflammatory agent, lipopolysaccharide (i.e., endotoxin derived from Escherichia coli). Systemically administered endotoxin amplifies neonatal hypoxic-ischemic brain injury in rodent and sheep models; some of these deleterious effects are mediated, at least in part, by systemic mechanisms (e.g., hypotension, hypoglycemia) [Lehnardt et al., 2003].

Drugs that block microglial activation and cytokine release protect the brain from excitotoxic damage [Dommergues et al., 2003]. Minocycline is a tetracycline derivative that crosses the blood–brain barrier and has been postulated to confer anti-inflammatory effects on microglia, as well as modulate immune cell activation, and cytokine and NO release, while also decreasing apoptosis. In the neonatal brain, minocycline appears to block hypoxia-ischemia-induced tissue damage and caspase-3 activation in rodents when given immediately before or after injury, but results are inconsistent [Arvin et al., 2002; Fox et al., 2005; Cai et al., 2006]. Minocycline attenuates lipopolysaccharide-induced brain injury and improves neurobehavioral outcomes in P5 rats via inhibition of microglial activation with resultant decreases in IL-1β and TNFα [Fan et al., 2005]. Low- and high-dose regimens were effective in reducing short-term hypoxia-ischemia-induced inflammation, protecting developing oligodendrocytes [Cai et al., 2006] and myelin content in neonatal rats [Carty et al., 2008], but this effect was only transient in another study of neonatal rodent stroke [Fox et al., 2005]. Following stroke, it decreases postischemic brain inflammation via inhibition of 5-LOX and enzymatic activation. Delayed therapy was found to decrease TNFα and matrix metalloproteinase (MMP)-12, but efficacy was lost when treatment was extended for a week after stroke [Wasserman et al., 2007]. These effects also appear to be species-dependent, with an increase in injury in developing C57B1/6 mice [Tsuji et al., 2004].

Apoptosis

Apoptosis is critical for normal brain development, but it is also an important component of injury following neonatal hypoxia-ischemia and stroke [Northington et al., 2005]. Activation of intrinsic or extrinsic apoptotic pathways leads to cleavage and activation of caspase 3, which is maximally produced in the neonatal period [Hu et al., 2000]. Pro-apoptotic Bax is present in high concentrations during the first 2 postnatal weeks [Lok and Martin, 2002]. While necrosis plays a major role in early neuronal death in both the immature and mature brain [Northington et al., 2001], there appears to be some aspect of apoptosis within the first 24 hours following neonatal hypoxia-ischemia [Portera-Cailliau et al., 1997], with a spectrum of cell death present that may result in heterogenous responses to anti-apoptotic therapies [Northington et al., 2005]. It is likely that apoptosis is not the cause of most acute cell death, but rather of delayed phases of injury and neurodegeneration. This delayed apoptotic cell death likely relates to target deprivation and loss of neurotrophic support [Northington et al., 2005]. Specific and nonspecific inhibition of caspases or cysteine proteases, which are highly activated after hypoxia-ischemia, has also been attempted with some success [Feng et al., 2003; Han et al., 2002; Blomgren et al., 2001; Ostwald et al., 1993]. See Chapter 14 for more details.

Neuroprotection

In addition to manipulating some of the mechanisms described above, there has been a recent focus on using hypothermia, which may reduce injury at multiple steps. Although neuroprotective interventions consisting of pharmacologic antagonism of glutamate, free radicals, inflammatory mediators, and apoptosis have been successful to some degree in experimental neonatal and adult animal models of hypoxic-ischemic brain injury, none of these other strategies has been successfully translated into clinical practice. Perhaps it is time to consider syngeristic and combinatorial therapies with hypothermia.

A recent therapy that is becoming standard of care for selected newborns is the use of therapeutic hypothermia for brain injury. It is postulated to work by modifying apoptosis and interrupting early necrosis [Edwards et al., 1995], reducing cerebral metabolic rate and the release of excitotoxins, NO, and free radicals [Globus et al., 1995]. Multiple animal models of perinatal brain injury demonstrate histological and functional benefit of early initiation of hypothermia [Laptook et al., 1994, 1997; Thoresen et al., 1995; Gunn et al., 1997; Gunn et al., 1998b]. Brief hypothermia initiated early after injury provided partial neuroprotection [Laptook and Corbett, 2002; Towfighi et al., 1994], but prolonged moderate hypothermia to 32–34°C for 24–72 hours results in sustained improvement in behavioral performance in newborn and adult animals [Gunn et al., 1997, 1998b]. The only complications were transient reductions of heart rate and blood pressure [Thoresen and Whitelaw, 2000].

Review of human neonatal studies shows reduction in mortality and long-term neurodevelopmental disability at 12–24 months of age, with more pronounced effects in moderately encephalopathic infants [Gunn et al., 1998a; Eicher et al., 2005; Gluckman et al., 2005; Shankaran et al., 2005; Azzopardi et al., 2009]. Sustained protection does depend on the degree of hypothermia, with maximum benefit obtained with cooling to 33–34°C, as well as in minimizing delay time to treatment [Bona et al., 1998; Gunn et al., 1997]. Mild hypothermia to this level appears to be well tolerated without serious adverse effects if initiated within the first 6 hours of life [Gunn et al., 1998a; Azzopardi et al., 2000; Thoresen, 2000; Shankaran et al., 2002]. In selective head cooling, treatment benefited infants with moderate but not severe amplitude integrated EEG changes, improving survival without severe neurodevelopmental deficits or an increase in complications [Gluckman et al., 2005]. In addition to severity of encephalopathy, larger infants were more responsive to hypothermia and at higher risk for injury if hyperthermic at any point [Wyatt et al., 2007]. In a second multicenter trial, whole-body cooling to 33.5°C, initiated within 6 hours and continued for 72 hours, resulted in less death and severe disability at 18 months. In a piglet model, the degree of hypothermia altered the amount of protection in the cortical gray matter relative to the deep gray matter [Iwata et al., 2005]. Whole-body cooling may be more effective in reducing temperature in the deep brain structures [Van Leeuwen et al., 2000]. Most recently, another whole-body cooling trial was completed (TOBY) and the results were consistent with the other two major trials, with a significant number of treated babies showing survival without neurodevelopmental sequelae (Figure 17-3) [Azzopardi et al., 2009].

Fig. 17-3 Summary of published trials of hypothermia for neuroprotection in the newborn.

CP, cerebral palsy.

Combinatorial therapy may provide more long-lasting neuroprotection, salvaging the brain from severe injury while also enhancing repair and regeneration, and hopefully providing additive, if not synergistic, protection.

Xenon is approved for use as a general anesthetic in Europe, and has shown promise as a protective agent. It is an NMDA antagonist, preventing progression of excitotoxic damage. It appears to be superior to other NMDA antagonists, possibly through inhibition of AMPA and kainite receptors, reduction of neurotransmitter release, or effects on other ion channels [Ma and Zhang, 2003; Dinse et al., 2005; Gruss et al., 2004]. The combination of xenon and hypothermia initiated 4 hours after neonatal hypoxia-ischemia provided synergistic histological and functional protection when evaluated at 30 days after injury in rodents [Ma et al., 2005]. More recently, an additive effect was shown after hypoxia-ischemia in P7 rats that were cooled to 32°C and received 50 percent xenon, with improvement in long-term histology and functional performance that exceeded the individual benefit of either [Hobbs et al., 2008]. Studies on xenon use in human neonates are under way.

N-acetylcysteine (NAC) is a medication, approved for neonates, that is a scavenger of oxygen radicals and restores intracellular glutathione levels, attenuating reperfusion injury and decreasing inflammation and NO production. Adding NAC therapy to systemic hypothermia reduced brain volume loss at both 2 and 4 weeks after neonatal rodent hypoxia-ischemia, with increased myelin expression and improved reflexes [Jatana et al., 2006]. Inhibition of inflammation with MK-801 has also been effective when combined with hypothermia in neonatal rats post hypoxic-ischemic injury [Alkan et al., 2001]. In P7 rats who underwent hypoxia-ischemia, followed by early topiramate and delayed hypothermia, improved short-term histology and function was seen [Liu et al., 2004]. This may provide a window for protection if hypothermia is delayed, which is possible, given the difficulty in initiation of cooling if infants are born outside hospital or transport is delayed.

Neurotrophic Factors

EPO is a 34-kDa glycoprotein that was originally identified for its role in erythropoiesis, but has since been found to have a variety of other roles. The pleiotropic functions of this cytokine include modulation of the inflammatory and immune responses [Villa et al., 2003], and vasogenic and pro-angiogenic effects through its interaction with vascular endothelial growth factor (VEGF) [Wang et al., 2004; Chong et al., 2002], as well as effects on CNS development and repair. EPO and EPO receptors are expressed by a variety of different cell types in the CNS, with changing patterns during development [Juul et al., 1999]. EPO plays a vital role in neural differentiation and neurogenesis early in development, promoting neurogenesis in vitro and in vivo [Shingo et al., 2001].

Recent evidence suggests that exogenously administered EPO has a protective effect in a variety of different models of immature brain injury [Sola et al., 2005b]. In newborn rodents, pretreatment with EPO before injury has a protective effect [Sola et al., 2005b]. In addition, postinjury treatment protocols have demonstrated both short- and long-term histological and behavioral improvement. A single dose of exogenous EPO administered immediately after neonatal hypoxic-ischemic injury in rats significantly reduced infarct volume and improved long-term spatial memory after injury [Kumral et al., 2004]. Single- and multiple-dose treatment regimens of EPO following neonatal focal ischemic stroke in rats also reduced infarct volume [Sola et al., 2005a] and improved short-term sensorimotor outcomes [Chang et al., 2005], but there may be more long-term behavioral benefit in female rats [Wen et al., 2006]. EPO treatment that was delayed 24 hours after neonatal hypoxia-ischemia also attenuated brain injury [Sun et al., 2005]. In addition, EPO enhances neurogenesis and directs multipotential neural stem cells toward a neuronal cell fate [Shingo et al., 2001; Wang et al., 2004; Gonzalez et al., 2007]. Following transient ischemic stroke, there is temporary precursor cell proliferation in the rodent subventricular zone (SVZ), with this precursor cell proliferation and differentiation favoring gliogenesis [Plane et al., 2004]. EPO has been shown to enhance neurogenesis in vivo in the SVZ following stroke in the adult rat [Wang et al., 2004]. Neurogenesis has also been demonstrated following EPO treatment with an increase in newly generated cells from precursors [Wang et al., 2004; Lu et al., 2005; Shingo et al., 2001], and possibly also an effect on cell fate commitment [Wang et al., 2004; Shingo et al., 2001] in vitro.

In humans, EPO is safely used for treatment of anemia in premature infants [Aher and Ohlsson, 2006]. EPO for neuroprotection is given in much higher doses (1000–5000 U/kg/dose) than that given for anemia [Chang et al., 2005; Demers et al., 2005; McPherson and Juul, 2007] to enable crossing of the blood–brain barrier, with unknown pharmacokinetics in humans. Recently, extremely low birth weight infants tolerated doses between 500 and 2500 U/kg/dose [Juul et al., 2008]. A recent trial of EPO for neonatal asphyxia showed that repeated low-dose therapy (300–500 U/kg every other day for 2 weeks) reduced the risk of disability for infants with moderate HIE and no negative hematopoietic side effects were observed [Zhu et al., 2009].

VEGF is a regulator of angiogenesis that also promotes neuronal cell proliferation and migration [Zachary, 2005]. The endothelial microenvironment establishes a vascular niche to promote survival and proliferation of progenitor cells, which is tightly coordinated with angiogenesis [Palmer et al., 2000]. VEGF-A is the most important member of a family of growth factors that also includes placental growth factor (PLGF) and VEGFs B, C, and D. VEGF-A expression occurs in cortical neurons during early development, switching to mature glial cells near vessels during later development. Following exposure to hypoxia, there is increased neuronal and glial expression [Krum and Rosenstein, 1998], directing vascularization and stimulating proliferation of astrocytes, microglia, and neuronal cell types [Forstreuter et al., 2002; Mu et al., 2003; Jin et al., 2002]. VEGF also has chemotactic effects on neurogenic zones in the brain [Yang and Cepko, 1996], increasing migration of stem cells during anoxia [Bagnard et al., 2001; Maurer et al., 2003]. VEGF knockout mice have severe impairments in vascularization, neuronal migration, and survival [Raab et al., 2004].

VEGF appears to play an essential role in the beneficial and protective effects of hypoxic preconditioning in neonatal mice [Laudenbach et al., 2007]. VEGF-R2 inhibition increases tissue injury and cell death in a neonatal stroke model, as well as reducing endothelial cell proliferation in the injured core [Shimotake, 2010].

Other trophic factors have also shown promise in reducing brain injury but, given their role in normal neurodevelopment, the effects of treatment are not known. Insulin-like growth factor-1 (IGF-1) has prosurvival properties that can prevent perinatal hypoxic or excitotoxic injury [Johnston et al., 1996; Pang et al., 2007]. Brain-derived neurotrophic factor (BDNF) is a neurotrophin that also provides neuroprotection in neonatal hypoxia-ischemia [Cheng et al., 1997, 1998; Holtzman et al., 1996; Husson et al., 2005]. It prevents spatial memory learning impairments after insult, but its effectiveness is limited by the stage of development [Husson et al., 2005; Cheng et al., 1998]. While protective in mice at P5, it exacerbates excitotoxicity at P0 and has no effect at later time points [Husson et al., 2005].

Stem Cells

Neural stem cells (NSCs) are multipotent precursors that self-renew and retain the ability to differentiate into a variety of neuronal and non-neuronal cell types in the CNS. They reside in neurogenic zones throughout life, such as the SVZ and subgranular zone of the dentate gyrus in rodent models, and help maintain cell turnover at baseline and replace injured cells by migrating to penumbral tissue after injury. NSC transplantation has shown potential as a therapeutic strategy in adult animal models of stroke and hypoxia-ischemia. Implanted cells integrate into injured tissue [Park et al., 2002], decreasing volume loss [Hoehn et al., 2002; Park et al., 2006a, b] and improving behavioral outcomes [Capone et al., 2007; Hicks et al., 2007]. In neonatal models, intraventricular implantation of NSCs after hypoxia-ischemia results in their migration to areas of injury [Park et al., 2006a, b]. These stem cells differentiate into neurons, astrocytes, and oligodendrocytes, as well as undifferentiated progenitors. These cells not only promote regeneration, but non-neuronal phenotypes inhibit inflammation and scar formation, while promoting angiogenesis and neuronal cell survival in both rodent and primate models [Imitola et al., 2004; Mueller et al., 2006]. While no adverse effects have been noted, efficacy is dependent on time of implantation, and the therapeutic window is not known. More recent technology enables labeling of stem cells, which can then be tracked from their site of implantation through their migratory path into the ischemic tissue [Modo et al., 2004; Guzman et al., 2007; Rice et al., 2007; Obenaus et al., 2007], which will make tracking of these cells in humans possible [Ashwal et al., 2009].