Pearlman 2004

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Hypoxicischemic brain injury in the term

infant-current concepts

Lina Shalaka, Jeffrey M. Perlmanb,*

aDepartment of Pediatrics, Southwestern Medical Center, University of Texas, 5323 Harry Hines Blvd.,

75390 Dallas, TX, United StatesbDepartment of Pediatrics, Weill Medical College of Cornell University, New York, NY 10021, United States

Accepted 4 June 2004

Abstract

An enhanced understanding of the cellular characteristics contributing to ongoing brain injury

following intrapartum hypoxia-ischemia has resulted in the implementation of targeted neuro-

protective strategies in the newborn period. This review briefly covers the pathogenesis of hypoxic-

ischemic injury with an emphasis on reperfusion injury; the role of magnetic resonance imaging in

the detection of such injury, and focuses on potential strategies both supportive and neuroprotectiveto prevent ongoing injury with a specific emphasis on modest hypothermia.

D 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Hypoxicischemic brain injury; Infant-current concepts cerebral palsy

1. Background

Hypoxicischemic cerebral injury that occurs during the perinatal period is one of the

most commonly recognized causes of severe, long-term neurological deficits in children,

often referred to as cerebral palsy (CP) [1,2]. The brain injury that develops is an evolvingprocess initiated during the insult and extends into a recovery period, the latter referred to as

the breperfusion phaseQ of injury. Clinically, it is the latter phase that is amendable to

potential intervention(s). Until recently, current management strategies have largely been

0378-3782/$ - see front matterD 2004 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.earlhumdev.2004.06.003

* Corresponding author.

Early Human Development 80 (2004) 125141www.elsevier.com/locate/earlhumdev

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supportive and not targeted toward the processes of ongoing injury [3]. Thus, it should not be

surprising that, despite improvements in perinatal practice during the past several decades,

the incidence of CP attributed to intrapartum asphyxia has remained essentially unchanged[1]. However, novel exciting strategies aimed at preventing ongoing injury are being

clinically evaluated and offer an opportunity for neuroprotection. In this discussion, we

briefly review the cellular characteristics of hypoxicischemic cerebral injury and the

current and future therapeutic strategies aimed at ameliorating ongoing brain injury

following intrapartum hypoxiaischemia.

2. Pathogenesis of hypoxicischemic cerebral injury

The principal pathogenetic mechanism underlying most of the neuropathology

attributed to intrapartum hypoxiaischemia is impaired cerebral blood flow (CBF). Thisis most likely to occur as a consequence of interruption in placental blood flow and gas

exchange, often referred to as basphyxiaQ or severe fetal acidemia. The latter is defined as a

fetal umbilical arterial pHV7.00 [1].

At the cellular level, the reduction in cerebral blood flow and oxygen delivery initiates a

cascade of deleterious biochemical events. Depletion of oxygen precludes oxidative

phosphorylation and results in a switch to anaerobic metabolism. This is an energy

inefficient state resulting in rapid depletion of high-energy phosphate reserves including

ATP, accumulation of lactic acid and the inability to maintain cellular functions [4].

Transcellular ion pump failure results in the intracellular accumulation of Na+, Ca+ and water

(cytotoxic edema). The membrane depolarization results in a release of excitatory

neurotransmitters and specifically glutamate from axon terminals. The glutamate then

activates specific cell surface receptors resulting in an influx of Na+ and Ca+ into

Fig. 1. Potential biochemical mechanisms of hypoxic ischemic cerebral injury.

L. Shalak, J.M. Perlman / Early Human Development 80 (2004) 125141126

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postsynaptic neurons. Within the cytoplasm, there is an accumulation of free fatty acids

secondary to increased membrane phospholipid turnover. The fatty acids undergo

peroxidation by oxygen free radicals that arise from reductive processes within mitochondriaand as byproducts in the synthesis of prostaglandins, xanthine and uric acid (see Fig. 1). Ca+

ions accumulate within the cytoplasm as a consequence of increased cellular influx as well as

decreased efflux across the plasma membrane combined with release from mitochondria and

endoplasmic reticulum. In selected neurons, the intracellular calcium induces the production

of nitric oxide (NO), a free radical that diffuses to adjacent cells susceptible to nitric oxide

toxicity. The combined effects of cellular energy failure, acidosis, glutamate release,

intracellular Ca+ accumulation, lipid peroxidation and nitric oxide neurotoxicity serve to

disrupt essential components of the cell with its ultimate death [2,5]. Many factors including

the duration or severity of the insult will influence the progression of cellular injury

following hypoxiaischemia (Table 1).

3. Delayed (secondary) brain damage

Following resuscitation, which may occur in utero or postnatally in the delivery room,

cerebral oxygenation and perfusion is restored. During this recovery phase, the concen-

trations of phosphorus metabolites and the intracellular pH return to baseline. However, the

process of cerebral energy failure recurs from 6 to 48 h later in a second phase of injury. This

phase is characterized by a decrease in the ratio of phosphocreatine/inorganic phosphate,

with an unchanged intracellular pH, stable cardiorespiratory status and contributes to further

brain injury [4,6]. In the human infant, the severity of the second energy failure is correlated

with adverse neurodevelopmental outcome at 1 and 4 years [7]. The mechanisms of

secondary energy failure may involve mitochondrial dysfunction secondary to extendedreactions from primary insults (e.g., calcium influx, excitatory neurotoxicity, oxygen free

radicals or nitric oxide formation). Indeed, mitochondria play a key role in determining the

fate of neurons following hypoxiaischemia. Thus, translocation of apoptotic triggering

proteins such as cytochrome c from the mitochondria to the cytoplasm can activate a cascade

of proteolytic enzymes termed caspases or cysteine proteases that eventually trigger nuclear

fragmentation [5]. Recent evidence also suggests that circulatory and endogenous

inflammatory cells/mediators also contribute to ongoing brain injury [8].

To discuss this process further, four basic mechanisms of ongoing injury are outlined

next.

3.1. Accumulation of cytosolic calcium

Calcium is an intracellular second messenger necessarily for numerous cellular reactions.

Under physiologic condition, cytosolic Ca+ concentration is strictly regulated at a very low

intracellular concentration. During hypoxiaischemia, there is an increase in Ca+ influx into

neuronal cells in part by stimulation of the NMDA receptors of the agonist-operated Ca+

channel by glutamate. In an opposite direction, Ca+ efflux across the plasma membrane is

interrupted by energy failure. Moreover, Ca+ is also released into cytoplasm from the

mitochondria by the Na+,H+-dependent antiport system and from the endoplasmic reticulum

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by inositol triphosphate (IP3) derived from the plasma membrane. These alterations of Ca+

balance result in increased intracellular Ca+, which interferes with many enzymatic reactions

including the activation of lipases, proteases, endonucleases and phospholipases and theformation of oxygen free radicals as byproducts of xanthine and prostaglandin synthesis.

Overall, the accumulation of cytosolic Ca+ after hypoxiaischemia has detrimental effects

on neuronal cells leading to irreversible brain damage [2,5].

3.2. Excitatory neurotransmitter release

Glutamate is a major excitatory amino acid within brain. The action of glutamate is

mediated by a number of receptor subtypes with the NMDA receptor predominating in

developing brain and is increased in areas of active development, e.g., striatum or

hippocampus. Within the NMDA receptor site, there are regions of agonist activity, i.e.,

glutamate, glycine as well as regions of antagonist activity, i.e., Mg

2+

, phencyclidine or PCP[2,5]. Each of these latter sites may offer the potential for neuroprotective strategies.

During hypoxiaischemia, glutamate accumulates within the synaptic cleft secondary to

increased release from axon terminals as well as impaired reuptake at presynaptic nerve

endings. The excessive glutamate acting on the receptor sites facilitates the intracellular

entry of Ca+ via the NMDA receptor-mediated channel and promotes the biochemical

cascade mentioned above leading to ultimate neuronal death [2,5].

3.3. Formation of free radicals

3.3.1. Oxygen free radicals

In aerobic cells, oxygen free radicals (i.e., O2, H2O2, OH

) are produced within the

cytoplasm and mitochondria. Under physiologic condition, oxygen free radicals aredestroyed rapidly by endogenous antioxidants (i.e., superoxide dismutase, endoperoxidase,

catalase) and scavengers (i.e., cholesterol, a-tocopherol, ascorbic acid, glutathione) [5,8].

During and following hypoxiaischemia, the generation of oxygen free radicals is increased

in excess of the protective capability. Two major sources of oxygen free radicals are the

byproducts of xanthine (derived from the breakdown of ATP) and prostaglandin synthesis

(derived from the breakdown of free fatty acids) (see Fig. 1). Oxygen free radicals contribute

to tissue injury by attacking the polyunsaturated fatty acid component of the cellular

membrane resulting in membrane fragmentation and cell death [5,8].

3.3.2. Nitric oxide

Nitric oxide (NO) is a weak free radical formed during the conversion ofl-arginine to l-citrulline by nitric oxide synthase (NOS). NOS is strongly activated during hypoxia

ischemia as well as during reperfusion and a large amount of NO is produced for extended

periods. NO when combined with superoxide generates a potent radical, peroxynitrite,

which activates lipid peroxidation. In addition, NO also enhances glutamate release [8].

3.3.3. Iron

Physiologically, iron (Fe) is maintained in non-toxic ferric state and bound to proteins

(i.e., ferritin, transferrin). During hypoxiaischemia, free ferric iron is released from these


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proteins and reacts with peroxides to generate potent hydroxyl radicals. In addition, free

ferric iron is reduced to a ferrous form, which further contributes to free radical injury [8].

3.4. Inflammatory mediators

Inflammatory mediators seem to play a critical role in the pathogenesis of hypoxic

ischemic brain injury. Thus expression of IL-1-h and TNF a messenger RNA has been

demonstrated within 14 h following hypoxia ischemia [9], along with the induction ofa

and h chemokines followed by neutrophil invasion of the area of infarction [10].

Furthermore, a low dose of bacterial endotoxin administered 4 h prior to hypoxiaischemia

in rats induces cerebral infarction in response to short periods of hypoxiaischemia that

usually causes little or no injury. This endotoxin induced sensitization of the immature brain

occurs independent of cerebral blood flow changes and/or hyperthermia and is associated

with an altered expression of CD14 mRNA [11]. Inflammatory cytokines may have a directtoxic effect via increased production of inducible nitric oxide synthase, cyclooxygenase and

free radical release, or indirectly via induction of glial cells to produce neurotoxic factors

such as excitatory amino acids. The important role of cytokines in perpetuating excitotoxic

injury is suggested from experimental observations in which administration of IL-1

endogenous receptor antagonists as well as neutralizing antibody are associated with a

reduction in excitotoxic and ischemic injury in mice [12,13]. However, cytokines that

potentiate brain damage following ischemia also seem to play a beneficial neurotrophic

effect. Thus, TNF knockout mice were shown to have increased neuronal cell degeneration

following ischemia and excitotoxic injury suggesting a critical role for endogenous TNF in

regulating the cellular responses to brain injury [14]. In addition, administration of

exogenous IL-6 following ischemia produced by middle artery occlusion also results in

marked neuroprotection [15]. Thus, inflammatory cytokines appear to exert both beneficialand deleterious effects following ischemia. This dual effect will likely complicate the task of

developing targeted interventions against the inflammatory response.

4. Mechanisms of neuronal cell death following hypoxiaischemia

The mechanism of neuronal cell death in animals and human following hypoxia

ischemia includes neuronal necrosis and apoptosis. The intensity of the initial insult may

determine the mode of death with severe injury resulting in necrosis, while milder insults

result in apoptosis [16].Necrosis is a passive process of cell swelling, disrupted cytoplasmic

organelles, loss of membrane integrity and eventual lysis of neuronal cells and activation ofan inflammatory process. By contrast, apoptosis is an active process distinguished from

necrosis by the presence of cell shrinkage, nuclear pyknosis, chromatin condensation and

genomic fragmentation, events that occur in the absence of an inflammatory response [17].

These two processes of neuronal death can be demonstrated following hypoxia ischemia in

both animals and humans [2,5,17]. Since the mechanisms of neuronal necrosis versus

apoptosis likely differ (see above), strategies to minimize brain damage in an affected infant

following hypoxiaischemia likely will have to include interventions that target both

processes.


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5. Detection of neuronal injury following hypoxiaischemia with magnetic resonance

(MR) imaging

MR techniques have become very important in delineating evolving structural,

metabolic and functional changes following intrapartum hypoxiaischemia [18]. Thus,

using conventional MR, three patterns of signal abnormalities can be identified including

injury to the thalami and/or posteriorlateral putamen with involvement of the subcortical

white matter in the most severe cases; injury to the parasagittal gray matter and subcortical

white matter, posterior usually more than anterior; and focal or multifocal injury [19].

Conventional MR studies are of limited value in the early phases, i.e., hours and days

following the insult. More recently, the use of advanced MR techniques, i.e., diffusion

weighted imaging (DWI), has been utilized to detect early injury [20]. This technique

measures the self-diffusion of water detected as an apparent diffusion coefficient (ADC)

[18]. Thus, the ADC decreases with acute injury and is reflective of reduced waterdiffusivity in the tissue. Studies of adult arterial infarcts have shown that DWI signal

changes occur within minutes of symptom onset and hours before changes become apparent

on T1- or T2-weighted images [21]. This earlier detection of injury may facilitate

intervention prior to irreversible injury. DWI changes may be negative if it is performed

earlier than 24 h of life or later than 8 days of life [20]. Recently, the development of

magnetic resonance spectroscopy (MRS) has facilitated the detection of tissue metabolism

and in vivo biochemistry [19,20]. Thus, proton MRS (1H-MRS) can identify a spectrum of

metabolites including lactate, creatine, N-acetylaspartate (NAA) and glutamine to name a

few. Indeed, elevated lactate levels have been demonstrated following hypoxiaischemia in1H-MRS with regional differences, i.e., higher lactate/NAA ratios in basal ganglia than in

occipito-parietal cortex [22]. Moreover, an early elevated lactate/creatine ratio with 1H-

MRS coupled with a low phosphocreatine/inorganic phosphate ratio done with phosphorusMRS correlated with neurodevelopmental outcome at 1 year [23]. Thus, the data indicate

that MRS plays an important role in the assessment of the encephalopathic infant following

intrapartum hypoxiaischemia.

6. Strategies to prevent ongoing injury following hypoxiaischemia

The goals of management of a newborn infant who has sustained a hypoxicischemic

insult and is at risk for evolving injury should include: (1) early identification of the infant

at highest risk for evolving injury, (2) supportive care to facilitate adequate perfusion and

nutrients to the brain and (3) consideration of interventions to ameliorate the processes ofongoing brain injury.

Each of these approaches is briefly discussed next.

7. Early identification of high risk infants

The initial step in management is early identification of those infants at greatest

risk for evolving to the syndrome of hypoxicischemic encephalopathy (HIE). This is


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a highly relevant issue because the therapeutic window, i.e., the time interval

following hypoxiaischemia during which interventions might be efficacious in

reducing the severity of ultimate brain injury, is likely to be short. Based onexperimental studies, it is estimated to vary from 2 to 6 h. Given this presumed short

window of opportunity, infants must be identified as soon as possible following

delivery in order to facilitate the implementation of early interventions. There are

increasing data to indicate that the highest risk infant can be identified shortly after

birth by a constellation of findings. These include evidence of a sentinel event during

labor, i.e., fetal heart rate abnormality, a severely depressed infant (low extended

Fig. 2. Amplitude EEG tracings depicting a normal pattern (top panel), moderate suppression (middle panel) and

severe suppression (bottom panel).


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Apgar score), the need for resuscitation in the delivery room (i.e., intubation, chest

compressionFepinephrine administration), evidence of severe fetal acidemia (cord

umbilical artery pHb7.00 and/or base deficit N

16 mEq/l) [24], followed by evidenceof an early abnormal neurologic examination and/or abnormal assessment of cerebral

function, i.e., integrated EEG [2527] (Fig. 2). Thus, in a prospective study of 50

infants who had evidence of intrapartum distress, Apgar score 5 at 5 min, or cord

arterial pH 7.00 and underwent an early neurologic examination using a modified

Sarnat staging system (stages 2 and 3 were regarded as abnormal) and a blinded

simultaneous a-EEG measurement, predictive values were calculated for a short-term

abnormal outcome defined as persistent moderate to severe encephalopathy beyond 5

days. An abnormal outcome evolved in 14 (28%) of the 50 infants. The neurologic

examination was performed at 5F3 h after delivery. A short-term abnormal outcome

occurred in 9 (53%) of 17 infants with initial stage 2 and in both infants with initial

stage 3 encephalopathy. In addition, 13 infants manifested features of both stages 1and 2 encephalopathy and post hoc were classified (S12). Three of the latter infants

(23%) developed an abnormal short-term outcome. The a-EEG was abnormal in 15

(30%) infants, 11 (73%) of whom developed an abnormal outcome. An abnormal a-

EEG was more specific (89% vs. 78%), had a greater positive predictive value (73%

vs. 58%), and had similar sensitivity (79% vs. 78%) and negative predictive value

(90% vs. 91%) when compared with an abnormal early neurologic examination. A

combination of abnormalities had the highest specificity (94%) and positive predictive

value (85%). Thus, a combination of the a-EEG and the neurologic examination

shortly after birth enhances the ability to identify high-risk infants and limits the

number of infants who would be falsely identified compared with either evaluation

alone [27].

8. Supportive care

Supportive care should be directed towards the maintenance of adequate ventilation,

avoidance of hypotension, judicious fluid management, avoidance of hypoglycemia and

treatment of seizures. For this review, we will focus on the avoidance of hypoglycemia and

the judicious treatment of seizures (Table 1).

9. Control of blood glucose concentrations

In the context of cerebral hypoxiaischemia, experimental studies suggest that both

hyperglycemia and hypoglycemia may accentuate brain damage. Regarding hyper-

glycemia, in adult experimental models as well as in humans, brain damage is

accentuated. However, in immature animals subjected to cerebral hypoxiaischemia,

hyperglycemia to blood glucose concentration of 600 mg/dl entirely prevents the

occurrence of brain damage [28]. Regarding hypoglycemia, the effects in experimental

animals vary and are related to the mechanisms of the hypoglycemia. Thus, insulin-

induced hypoglycemia is detrimental to immature rat brain subjected to hypoxia


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ischemia. However, if hypoglycemia is induced by fasting, a high degree of protection is

noted [29]. This protective effect is presumed to be secondary to the increased

concentrations of ketone bodies, which presumably serve as alternative substrates to the

immature brain. In a recent retrospective report, the potential contribution of initialhypoglycemia to the development of neonatal brain injury in term infants with severe

fetal acidemia was assessed in 185 term infants admitted to neonatal intensive care with

an umbilical arterial pHb7.00. Short-term neurologic outcome measures included death as

a consequence of severe encephalopathy and evidence of moderate to severe

encephalopathyFseizures. Hypoglycemia was defined as a blood glucose V40 mg/dl.

Forty-one (22%) infants developed an abnormal neurologic outcome including 14 (34%)

with severe HIE who died, 24 (59%) with moderate to severe HIE and 3 (7%) with

seizures. Twenty-seven (14.5%) of the 185 infants had an initial blood sugar V40mg/dl

(Table 2). An abnormal neurologic outcome was noted in 15/27 (56%) infants with a

blood sugarV40 mg/dl versus 26/158 (16%) of infants with a blood sugar N40 mg/dl had

(p=0.00003) (odds ratio (OR) 6.3 (95% CI 2.6, 15). By multivariate logistic analysis,four variables were significantly associated with abnormal outcome. However, an initial

blood glucose V40 versus N40 mg/dl (p=0.001) (OR=18.5, 95% CI=3.1111.9) was the

most significant variable. The other parameters, i.e., cord arterial pHV6.90 versus N6.90

(p=0.003), OR=9.8 (CI=2.144.7), a 5-min Apgar score V5 versus N5 (p=0.006),

OR=6.4 (CI=1.724.5) and the requirement for intubationFCPR versus neither (p=0.02),

OR=4. 7 (CI=1.217.9) were less so [30]. These data suggest that initial hypoglycemia is

an important risk factor for perinatal brain injury particularly in depressed term infants

requiring resuscitation with severe fetal acidemia. Thus, in the ongoing management of

Table 1

A guide to the supportive management of an infant at risk for hypoxicischemic cerebral injury

VentilationMaintain paCO2 within a normal range

Perfusion

Promptly treat hypotension

Avoid hypertension

Fluid status

Initial fluid restriction

Follow serum sodium and daily weights

Blood glucose

Maintain blood glucose within a normal range

Avoid hypoglycemia

Seizures

Treat clinical seizures, particularly with an electrographic correlate

Potential role of prophylactic phenobarbital

Electrolyte imbalance

Monitor serum electrolytes, calcium and magnesium


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hypoxiaischemia, a glucose level should be screened shortly after birth and monitored

closely. If the blood glucose is low, it should be promptly corrected.

9.1. Seizures

Hypoxicischemic cerebral injury is the most common cause of early onset neonatal

seizures [2]. Although seizures are a consequence of the underlying brain injury, seizures

activity in itself may contribute to ongoing injury. Experimental evidence strongly

suggests that repetitive seizures disturb brain growth and development as well as increase

the risk for subsequent epilepsy [31,32]. Despite the potential adverse effects of seizures,

the question of which infants should be treated remains controversial [2,33]. This is a part

related to the observation that not all clinical seizures have an electrographic correlation

[34].

9.2. Prophylactic phenobarbital

Experimental data indicates that barbiturate pretreatment and even early posttreatment in

adult animals subjected to cerebral hypoxiaischemia reduces the severity of ultimate brain

damage. The prophylactic administration of bhigh doseQ barbiturates to infants at highest

risk for evolving to HIE has been evaluated in two small studies with conflicting results

[35,36]. In the first randomized study, the administration of thiopental initiated within 2 h

and infused for 24 h did not alter the frequency of seizures, intracranial pressure or short-

term neurodevelopmental outcome at 12 months [35]. Of importance was the observation

that systemic hypotension occurred significantly more often in the treated group. In thesecond randomized study, phenobarbital 40 mg/kg body weight administered intravenously

between 1 and 6 h to asphyxiated infants was associated with subsequent neuroprotection

[36]. Although there was no difference in the frequency of seizures between the two groups

in the neonatal period, 73% of the pre-treated infants compared to 18% of the control group

(pb0.05) revealed normal neurodevelopmental outcome at 3-year follow-up. No adverse

effect of phenobarbital administration was observed in this study. Thus, the role of

prophylactic barbiturates in high-risk infants remains unclear at this current time and may

be worthy of further investigation.

Table 2

Characteristics of infants with abnormal as compared to normal outcome with initial hypoglycemia

Characteristic Abnormal outcome n=15 Normal outcome n=12 p-valueCPR 7 1 0.08

IntubationFCPR 12 4 0.04

5-min Apgar scoreb5 11 1 0.001

Cord arterial pH 6.86F0.07 6.92F0.04 0.004

Base deficit 19F4 18F2

Initial blood pressure (mm Hg) 34F10 42F10 0.006

Initial blood sugar (mg/dl) 18F15 17F11

Initial temperature (8C) 36.3F0.7 36.7F0.9

CPR=cardiopulmonary resuscitation.


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10. Potential neuroprotective strategies aimed at ameliorating secondary brain injury

10.1. Hypothermia

Modest systemic or selective cooling of the brain by as little as 24 8C has been shown

to reduce the extent of tissue injury in experimental studies as well as human following

events such as stroke, trauma or cardiac arrest [3753]. Potential mechanisms of

neuroprotection with hypothermia include inhibition of glutamate release, reduction of

cerebral metabolism which in turn preserves high energy phosphates, decrease in

intracellular acidosis and lactic acid accumulation, preservation of endogenous antiox-

idants, reduction of nitric oxide production, prevention of protein kinase inhibition,

improvement of protein synthesis, reduction of leukotriene production, prevention of

blood brain barrier disruption and brain edema and inhibition of apoptosis [47,49].

Although neuroprotection following intra-ischemic hypothermia is well established, theeffects of post-ischemic hypothermia have been less certain. The latter is the typical

scenario in the human newborn. Before initiating hypothermia as a therapeutic strategy in

neonates, several factors had to be considered. The first relates to duration of therapy.

Thus, brief post-ischemic hypothermia, i.e., less than 6 h results, is only partial or

temporary neuroprotection in most experimental animals [37,49]. Prolonging post-

ischemic hypothermia from 24 to 72 h attenuates brain damage and improves behavioral

performance [41,42,49]. The second factor relates to the degree of hypothermia.

Accordingly, moderate hypothermia at brain temperatures of 3234 8C initiated

immediately or within a few hours after reperfusion and continued for 2472 h has been

shown to favorably affect outcome in adult and newborn animals [41,42]. The third factor

relates to the delay in initiation of therapy. Clearly, the sooner hypothermia can be

initiated, the more likely it is to be successful. Indeed, studies indicate that, if therapy isinitiated beyond 6 h following hypoxiaischemia or following the onset of seizures, that

hypothermia is not neuroprotective [43,44]. The fourth factor relates to the method of

achieving hypothermia. Selective head cooling is attractive because it reduces potential

side effects (see below) but is associated with differential gradients within the brain [54].

Total body cooling is more likely to be associated with side effects but less temperature

gradient within the brain. A major concern of hypothermia in newborn infants relates to

potential adverse effects including hypoglycemia, reduction of myocardial contractility

and ventilationperfusion mismatch, increased blood viscosity, acidbase and electrolyte

imbalance and an increased risk of infection [49,55].

To address the practicality and safety of mild or minimal hypothermia, pilot studies

were conducted utilizing both selective and systemic methods to cool the brain in termneonates with moderate to severe encephalopathy [5557]. Subsequently, two large

randomized multicenter studies have been undertaken and completed in term infants at

highest risk for evolving to moderate and/or severe encephalopathy, using either

selective or systemic modest hypothermia. One of these studies utilizing selective head

cooling has reported follow up results at 18 months (Alistair Gunn-personal

communication). No effect of hypothermia with regard to the development of cerebral

palsy and/or death was observed for all infants treated. However, for infants who

presented with moderate encephalopathy and without electrographic seizures, a


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significant benefit of hypothermia was noted. No effect was observed in those infants

with severe encephalopathy and/or early seizures. Several critical questions need to be

addressed as a consequence of these preliminary data: (1) What is the idealtemperature for selective head cooling? (2) Which is the best mode of inducing

hypothermia, i.e., selective head versus total body cooling? (3) How long should

hypothermia be maintained? (4) Will a multimodal approach especially for the more

severely affected infants be more effective than hypothermia alone?

10.2. Oxygen free radical inhibitors and scavengers

One therapeutic approach for the destruction of oxygen free radicals generated during

and following hypoxiaischemia is the administration of specific enzymes known to

degrade highly reactive radicals to a non-reactive component. Superoxide dismutase and

catalase are antioxidant enzymes conjugated to polyethylene glycol that prolongs theircirculatory half life and facilitates penetration across the blood brain barrier [8]. Because

of their large molecular sizes, they are restricted to the vascular space. Thus, the positive

effects of these conjugated compounds are presumably from within cerebral vasculature by

improving cerebral blood flow [8]. In newborn animals, neuroprotection has only been

shown when these agents have been administered several hours prior to the hypoxic

ischemic insult [3,58].

A second group of free radical inhibitors that have been shown to be effective in

experimental animals are agents that inhibit specific reactions in the production of

xanthines. Thus, both allopurinol and oxypurinol, xanthine oxidase inhibitors, protected

the immature rat from hypoxicischemic brain damage when the drugs were administered

early during the recovery phase following resuscitation [3]. In a recent clinical study,

allopurinol administered to asphyxiated infants reduced blood concentrations of oxygenfree radicals when compared to control infants [59].

A third group of free radical inhibitors has targeted the formation of free radicals

specifically hydroxyl radical from free iron during reperfusion [8]. Thus, desferoxamine, a

chelating agent, prevents the formation of free radical from iron, reduces the severity of

brain injury and improves cerebral metabolism in animal models of hypoxiaischemia

when given during reperfusion [8,60].

Finally, lazeroids which are nonglucocorticoid, 21-aminosteroids, preventing iron-

dependent lipid peroxidation by scavenging peroxyl radicals have been shown to be

neuroprotective in both adult and immature models of hypoxicischemic brain damage [3].

10.3. Excitatory amino acid antagonists

Given the important role of excessive stimulation of neuronal surface receptors by

glutamate in promoting a cascade of events leading to cellular death [2,5], it has been

logical to identify pharmacological agents that would either inhibit glutamate release or

block its postsynaptic action.

Glutamate receptor antagonists (i.e., NMDA, AMPA subtypes) have been extensively

investigated in experimental animals. Non-competitive antagonists provided a reduction in

brain damage in adult animals even when administered up to 24 h after the insult. The


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available NMDA antagonists include dizocilpine (MK-801), magnesium, phencyclidine

(PCP), dextrometorphan and ketamine [3].

MK-801 has been shown to be an efficacious agent in reducing the extent of hypoxicischemic brain damage in both adult and neonatal animal models [3]. This neuroprotective

effect appears to be greater in immature compared to the adult model. This age-related

neuroprotective effect may reflect the alterations of density and distribution of glutamate

receptor subtypes during development of the brain.

Magnesium is an NMDA antagonist blocking neuronal influx of Ca+ within the ion

channel. The effect of magnesium also appears to be dependent on the maturation of the

glutamate receptor system. Thus, in a developmental study in mice, excitotoxic neuronal

death was limited by magnesium which was effective only after development of several

aspects of the excitotoxic cascade, including the coupling of the calcium influx follo wing

NMDA over stimulation, and the presence of magnesium-dependent calcium channels [61].

The potential neuroprotective effect of magnesium has revealed conflicting data inexperimental neonatal studies. Thus, when administered shortly following an intra-

cerebral injection of NMDA, magnesium sulfate reduced brain injury in newborn rat.

However, other neonatal studies have failed to demonstrate neuroprotection. In a

piglet model of asphyxia, magnesium sulfate administered 1 h following resuscitation

failed to decrease the severity of delayed energy failure. In addition, in a near-term

fetal lamb model, magnesium sulfate administered before and during umbilical cord

occlusion did not influence the electrophysiologic responses or neuronal loss

[3,62,63].

Magnesium sulfate is an attractive agent because of its frequent use in the United State.

It is often administered to mothers as a tocolytic agent or to prevent seizures in mothers

with pregnancy-induced hypertension. Preliminary retrospective observation in premature

infants noted a reduced incidence of CP at 3 years in those exposed to antenatalmagnesium sulfate [64]. A small randomized clinical study showed some subtle benefits,

i.e., on CT imaging and improved feeding at 14 days from magnesium infusion [65].

Clearly, future research is necessary in order to determine the potential neuroprotective

role of magnesium.

10.4. Prevention of nitric oxide formation

The inhibition of NOS is another potential treatment modality to decrease brain injury.

The experimental effects of NOS inhibitors vary and are animal species specific and

influenced by the pharmacological agent used, the dose and timing of therapy. Thus, in

adult experiments, the severity of neuronal loss can be reduced by the prior administrationof inhibitors of NOS activity. A similar protective effect has been observed in the

immature rat, whereas in fetal sheep neuronal injury appears to be accentuated following

hypoxiaischemia [3]. Further studies are clearly necessary.

10.5. Calcium channel blocks

The elevation of cytosolic calcium during hypoxicischemic injury makes the reduction

of intracellular accumulation a highly desirable therapy. One strategy of preventing


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calcium toxicity is to inhibit Ca+ influx into neurons by using calcium channel blocks

(e.g., flunarizine, nimodipine, nicardipine). Flunarizine has been studied in fetus and

newborn animals and the neuroprotective effect has only been demonstrated when givenprophylactically but not following hypoxicischemic insults [3]. Nicardipine was tested in

four severely asphyxiated infants but the positive effect was counteracted by the adverse

effects of significant hemodynamic disturbance [66]. Indeed, current calcium channel

blocks are in general contraindicated in the neonate and young infant in the United States

because of significant adverse cardiovascular effects.

10.6. Other studied therapies in immature animals

Other avenues of potential neuroprotection include platelet-activating factor antagonists

[67], adenosinergic agents [68], monosialoganglioside GM1 [69], growth factors, e.g.,

nerve growth factor (NGF) [70], insulin growth factor-1 [71] and blocking the apoptoticpathways, i.e., minocycline [72], erythropoeitin, etc. [73,74].

11. Conclusions

Much progress has been made toward understanding the mechanisms contributing to

ongoing brain injury following hypoxiaischemia. This should facilitate more specific

pharmacologic intervention strategies that might provide neuroprotection during the

reperfusion phase of injury. Early identification of infants at highest risk for brain injury

is critical if such interventions are to be successful. Moreover, given the small number of

asphyxiated full-term infants that are likely to be admitted to any one neonatal intensive

care unit over an extended time period implores a continued multicenter treatmentapproach to allow inclusion of an adequate number of infants to achieve statistical

validity.

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