Hypoxic ischaemic encephalopathy

SYMPOSIUM: NEONATOLOGY

Hypoxic ischaemicencephalopathyAnitha James

Vaishali Patel

Abstract
Over the past several years, basic and clinical research has improved our
understanding of critical cellular and molecular events which eventually

lead to brain damage following perinatal hypoxia-ischaemia. The knowl-

edge that perinatal hypoxia-ischaemia is a process that evolves over

hours to days provides a “window of opportunity” for intervention. This

review briefly covers the biochemical and physiological changes that

occur in the neonatal brain following hypoxia-ischaemia.

Keywords encephalopathy; hypoxia; ischaemia; pathophysiology;

perinatal

Introduction

Perinatal hypoxia-ischaemia (HI) is an important cause of brain

injury in the newborn and can result in long-term neurological

deficits. Even in the developed world, hypoxic ischaemic en-

cephalopathy (HIE) is common affecting approximately 1.5e2

per 1000 live born term infants. The pathophysiology of hypoxic

ischaemic brain injury in the term infant is multifactorial and

complex. It is becoming evident that HI is the final common

pathway for a complex convergence of events, some genetically

determined and some triggered by in utero stress.

Reduced cardiac output in the setting of hypoxia is referred to

as hypoxia-ischaemia. Volpe defines hypoxaemia as the

“diminished amount of oxygen in the blood supply” and cerebral

ischaemia as the “diminished amount of blood perfusing the

brain” which compromises both oxygen and substrate delivery to

the brain. In moderate to severe insult, the initial ischaemia is

followed by hyperaemia leading to reperfusion injury to the brain

after a latent period. The immature brain is more resistant to

injury from HI events because of lower cerebral metabolic rate,

plasticity of immature central nervous system, immaturity in the

development of balance in the functional neurotransmitters.

However the brain can become vulnerable to severe hypoxaemia

when cerebral blood flow is reduced beyond a critical threshold.

Cellular effects

Effects of HI on cellular energy metabolism

At the cellular level, the reduction in cerebral blood flow with

reduced substrate (oxygen and glucose) delivery to the brain

Anitha James MD MRCPCH is a Consultant Neonatologist in the

Department of Child Health, Royal Gwent Hospital, Newport Wales, UK.

Conflicts of interest: none.

Vaishali Patel MD MRCPCH is a Specialist Neonatal Registrar in the

Department of Child Health, Royal Gwent Hospital, Newport Wales, UK.

Conflicts of interest: none.

PAEDIATRICS AND CHILD HEALTH --:- 1

Please cite this article in press as: James A, Patel V, Hypoxic ischaemic en10.1016/j.paed.2014.02.003

initiates a series of biochemical events. Cellular energy becomes

dependent on anaerobic metabolism which is an energy ineffi-

cient state resulting in rapid depletion of high energy phosphate

reserves including adenosine triphosphate (ATP) and phospho-

creatine, accumulation of lactic acid and impaired cellular

homoeostasis. Depletion of high energy phosphates initiates the

cascade of events leading to neuronal death after HI insult. Loss

of cellular ATP compromises those metabolic processes that

require energy for their completion such as NaþeKþ pump which

normally causes Na extrusion through the plasma membrane in

exchange for potassium. Transcellular ion pump failure results in

the intracellular accumulation of Naþ, Cl� and water (cytotoxic

oedema). Intracellular Naþ, Cl� and water will continue to

accumulate with the resultant decrease in ionic gradients and

widespread depolarisation.

Neuronal injury

The pathophysiology of brain injury secondary to HI is associated

with two phases, primary and secondary energy failure, based on

characteristics of the cerebral energy state documented in both

preclinical models and human infants.

During initial phase of HI, there is a marked decline in high

energy phosphate level and marked increase in cerebral lactate

level (primary energy failure) leading to failure of NaþeKþ

pump and depolarisation of cell. The membrane depolarisation

results in the release of excitatory neurotransmitters like gluta-

mate. There is also cytoplasmic accumulation of calcium with

activation of a variety of calcium mediated enzymes. Many fac-

tors like duration and severity of insult influence the progression

of cellular injury.

Following reperfusion, cerebral perfusion and oxygenation

are restored to near normal and concentration of high energy

phosphates and intracellular pH return to baseline (latent

period).

A second decline in high energy phosphate levels occurs from

6 to 48 hours after the initial insult (secondary energy failure).

This is characterised by mitochondrial dysfunction leading to

energy failure secondary to extended reactions from primary

insults. Secondary energy failure differs from primary energy

failure as the decline in the levels of high energy phosphate and

increase in cerebral lactate are not accompanied by brain

acidosis. The pathogenesis of secondary energy failure involves

continuation of the excitotoxic-oxidation cascade, apoptosis,

inflammation, altered growth factor levels, and protein synthesis.

The severity of secondary energy failure is correlated with

adverse neurodevelopmental outcome at 4 years.

Current therapies are aimed at preventing injury during this

reperfusion period where infant is at risk of continued injury

(Figure 1).

Biochemical cascades

Accumulation of cytosolic calcium

Accumulation of intracellular calcium (Ca2þ) plays an important

role in the mediation of cell death in HI. Calcium is an important

second messenger in brain metabolism. The concentration of

calcium is tightly regulated with almost all calcium tightly bound

within subcellular organelles like mitochondria and endoplasmic

� 2014 Published by Elsevier Ltd.

cephalopathy, Paediatrics and Child Health (2014), http://dx.doi.org/

http://dx.doi.org/10.1016/j.paed.2014.02.003

Hypoxia-ischaemia

Anaerobic metabolism

ATP

Glutamate uptake Ca+2

Ca+2

Cell death

Free radicals Cytoskeletaldisruption

Membrane depolarisation

Glutamate release Glutamate

Xanthine oxidaseNitric oxide synthetase

Lipases

ProteasesMicrotubule disassembly

Nucleases

Membrane injury

Activationof caspases

Figure 1 Pathogenesis of hypoxic ischaemic encephalopathy.


reticulum, with very low concentration of free cytosolic calcium.

HI increases the free cytosolic concentration of calcium due to

� calcium influx into the cell by the glutamate induced

stimulation of NMDA receptors

� release of sequestrated stores from the mitochondria and

endoplasmic reticulum

� disruption of Ca2þ efflux through the cellular membrane

due to energy failure

The increased intracellular Ca2þ in turn activates numerous

enzymes like lipases, proteases, phospholipase C, endonucleases

which all affect the structural integrity of the cell. It also causes

uncoupling of oxidative phosphorylation in mitochondria and

formation of reactive oxygen species. In selected neurons it in-

duces the production of nitric oxide (NO) which diffuses into the

adjacent cells. The excessive intracellular calcium accumulation

causes membrane disintegration and neuronal death.

Excitatory amino acids (EAA)

Excessive stimulation of glutamate receptors appears to play an

important role in the pathogenesis of neonatal brain injury

caused by HI. Glutamate is the major excitatory transmitter in the

human brain. Glutamate is not degraded, but instead is removed

from the synaptic cleft by energy dependent neuronal and glial

uptake transporters. The action of glutamate is mediated by

NMDA (N-methyl-D-aspartate) and AMPA (alpha-amino-3-



hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. NMDA

receptors predominate in the developing brain.

During HI, there is accumulation of glutamate in the synaptic

cleft secondary to increased release from the axon terminal as

well as impaired uptake secondary to energy failure. This leads to

overstimulation of post-synaptic EAA receptors especially NMDA

receptors causing depolarisation of cellular membrane which

facilitates the intracellular entry of sodium and calcium pro-

moting the biochemical cascade leading to ultimate neuronal

death. The increased vulnerability of certain regions of brain to

HI injury is related to the increased expression of glutamate

receptors.

Oxidative stress

Oxidative stress is a term used for the increase in free radical

production as a result of oxidative metabolism under patholog-

ical condition. The concept of ischaemia followed by reperfusion

is important to the understanding of oxidative stress. At the

normal physiological state, more than 80% of oxygen in the cell

is reduced to energy equivalents (ATP) and the rest is converted

to superoxide anions which are scavenged enzymatically by su-

peroxide dismutase, catalase, glutathione peroxidise and non-

enzymatically by reaction with antioxidant molecules such as

alpha-tocopherol and ascorbic acid. During reperfusion, when

there is adequate oxygenation in the cells damaged by hypoxia,

mitochondrial oxidative phosphorylation is overwhelmed and

reactive oxygen species accumulate. Antioxidant defences get

depleted and free radicals damage the cells by peroxidation of

lipid membrane, alteration of membrane potential, activation of

proapoptotic mediators and direct DNA and protein damage.

The brain is particularly susceptible to free radical attack and

lipid peroxidation because of its high lipid content specifically

polyunsaturated fatty acid (PUFA). This vulnerability is

increased in term newborn brain as PUFA content of the brain

increases with gestation, newborn brain has underdeveloped

antioxidant enzymes and the newborn brain is rich in free iron

which can catalyse the production of various reactive oxygen

species.

Reactive nitrogen species (RNS)

Nitric Oxide (NO), a water soluble diffusible gas can contribute to

tissue injury. NO is generated by three distinct NO synthases:

neuronal (nNOS), endothelial (eNOS) and inducible (iNOS).

nNOS and eNOS are activated by increased intracellular calcium

whereas iNOS is upregulated by hypoxia, cytokines and is cal-

cium independent. nNOS contributes to NO production during

ischaemia and reperfusion, but iNOS mainly contributes to NO

production during reperfusion. Cerebral ischaemia stimulates

production of NO by neurons and microglia. Many of the adverse

effects of NO are mediated by its interaction with oxygen free

radical superoxide (O2�) to produce highly reactive and toxic

peroxynitrite (ONOOe) which activates lipid peroxidation. NO

also enhances glutamate release.

Inflammatory mediators

Inflammation plays an important role in pathogenesis of HI brain

injury. Several studies have shown a relationship between

maternal infection and neonatal brain injury. Lipopolysaccharide

sensitises the perinatal brain to HI and can worsen injury. It has





been proposed that cytokines may be the final common media-

tors of brain injury that is initiated by HI, reperfusion and

infection. The cytokines implicated in the brain injury associated

with HI are IL-1b, TNF a, IL-6, IL-8, PAF (platelet activating

factor), arachidonic acid and its metabolites. They act on

different cells like neurons, astrocytes, microglia and endothe-

lium. The cytokines are produced systematically by the mother

or fetus and affect the brain through vascular mechanism or by

entry across the bloodebrain barrier (BBB) and direct action on

brain parenchyma. There is also neutrophil accumulation in

brain blood vessels which may contribute to neuronal injury by

obstructing micro vascular flow or by release of free radicals.

Vascular effects

Cerebrovascular autoregulation

Cerebrovascular autoregulation describes the ability of the cere-

bral arteries to adjust their level of resistance as systemic blood

pressure fluctuates to maintain cerebral blood flow (CBF) within

an established range. In normal physiological condition CBF is

kept constant within a range of cerebral perfusion pressures

preventing swings of CBF sustained for longer than 10e20 sec-

onds. This is mediated by interplay between endothelial derived

constricting and relaxing factors. Studies of cerebral blood flow

indicate that blood flows are the highest in cerebral grey matter,

the nuclear structures of brain stem and the diencephalon. They

are lowest in the cerebral white matter.

Newborns with HI injury are at risk of CBF dysregulation.

With the loss of cerebral autoregulation, the CBF becomes

pressure passive. This is characterised by CBF becoming directly

dependent on systemic arterial blood pressure, with any decline

in blood pressure placing the infant at increased risk for ischae-

mic brain injury. Alteration in arterial PaO2 and PaCO2, acidosis,

adenosine and prostaglandins, NO are important regulators of

cerebral blood flow in the perinatal period.

In moderate asphyxia it has been shown that cerebral autor-

egulation is disturbed, but cerebral CO2 vasoreactivity is intact.

Loss of CO2 vasoreactivity has been found only in severe asphyxia.

During and following HI, neonatal brain is at a risk of cerebral

ischaemia secondary to loss of autoregulation and the development

of pressure passive cerebral circulation. Following the initial

ischaemia, a delayed and sustained increase inCBFdevelopswhich

can be shown by Doppler ultra sound and near-infrared spectros-

copy (NIRS). The Doppler shows increase in mean flow velocity

with decreased resistance index confirming vasodilatation and

NIRS shows the loss of vascular reactivity and an increase in CBF.

Genetic

The wide variability in the effect of HI on newborn brain high-

lights the probability that genetic factors play a significant role.

Gender difference with respect to the response to HI has also

been observed. Du and colleagues reported that male and female

rodent neurons grown in separate cultures differ in their activa-

tion of cell death pathway. Male neurons in culture are more

sensitive to death from exposure to NMDA and NO whereas fe-

male neurons were preferentially sensitive to caspase 3 inhibi-

tion. These sexually dimorphic differences in cell death pathway

might contribute to higher incidence of cerebral palsy in boys

than in girls.



Forms of cell death: necrosis and apoptosis

HI insult leads to cell death by necrosis or apoptosis or both

depending on the severity of the insult and the maturational state

of the cell. The more intense and long-lasting the HI insult, the

greater is the number of neuronal and glial cells which die.

Necrotic cell death occurs typically after severe relatively brief

insults. Apoptosis mediated by programmed cell death on the

other hand occurs after moderate, longer acting insults.

Apoptotic neuronal death predominates among immature neu-

rons, whereas necrotic cell death among mature neurons.

Though the cell death process have been generally classified

into distinct categories of necrosis and apoptosis, these distinc-

tions are now being replaced with a much more nuanced un-

derstanding of the overlap and interaction of common

mechanisms shared by various forms of cell death.

Often early cell death during ‘ischemic phase’ appears

necrotic. Necrosis is a passive process characterised by

biochemical events resulting from membrane ion pump failure

resulting in cell swelling, dispersed chromatin, loss of membrane

integrity and eventual lysis of neuronal cells causing inflamma-

tion and phagocytosis.

Later cell death during ‘reperfusion phase’ is dominated by

apoptosis. Apoptosis is an active process and is characterised by

cell shrinkage, chromatin condensation, intact membranes and

cell death. It occurs due to activation of caspases (cysteine pro-

teases). Caspase-3 activated within 1e3 hours after neonatal HI

is a principal trigger of apoptosis. Other factors involved in

apoptosis are an imbalance of anti-apoptotic and pro-apoptotic

proteins, extracellular accumulation of glutamate, increased

cytosolic Ca2þ, activation of specific death genes i.e. Bax-gene by

free radicals and lack of neuroprotective factors such as neuro-

trophines and oligotrophines. The time course of apoptotic death

in HI is slower than that of necrotic death which provides a more

prolonged window of opportunity for therapeutic intervention.

Cells showing morphology intermediate between that of

classic apoptosis and necrosis, referred to as hybrid cells have

been described. The nuclei of such cells have large, irregularly

shaped chromatin clumps like apoptotic neurons, but the cyto-

plasm shows changes similar to necrotic neurons.

Neuropathology

Although the neuropathological features of neonatal HIE vary

with the gestational age, the nature of the insult and the types of

interventions, certain basic lesions can be recognised.

‘Selective neuronal necrosis’ is the most common variety of

injury in neonatal HIE referring to neuronal necrosis in a char-

acteristic often widespread distribution. The topography depends

mainly on severity and temporal characteristics of the HI insult

and also on gestational age. Based on correlative clinical and

brain imaging findings, three basic patterns of selective neuronal

necrosis have been described in term newborns with HIE.

However, there can be considerable overlap.

The regional distribution of the excitatory glutamate receptors

i.e. NMDA and AMPA has been described as the single most

important determinant of the distribution of selective neuronal

necrosis with maximal neuronal injury occurring where the

expression of these receptors is the greatest. Enhanced vulnera-

bility to attack by ROS and RNS also plays an important role.




Three basic patterns of selective neuronal necrosis

Basic pattern Topography Severity and timing of HI insult

Diffuse neuronal injury Cerebral cortex, thalamus, basal ganglia, brain

stem, cerebellum, spinal cord

Very severe, very prolonged

Cerebral cortical e deep nuclear injury Cerebral cortex, basal ganglia, thalamus Moderate to severe, gradual

Deep nuclear e brain stem injury Basal ganglia, thalamus, brain stem Severe, relatively abrupt

Table 1


Cerebral ischaemia with impaired cerebrovascular autor-

egulation with pressure passive cerebral circulation and systemic

hypotension, regional variation in vascular and metabolic factors

also play a role.

� Diffuse neuronal injury: occurs with very severe and very

prolonged insults. Neurons of the cerebral cortex are

particularly vulnerable, mainly the hippocampus. Also the

neurons in the paracentral, perirolandic cortex and visual

cortex are affected. Cerebral white matter, especially in

parasagittal, watershed distributions, also may be affected,

often in association with involvement of overlying cortex.

In addition, neurons in the thalamus, basal ganglia, brain

stem and cranial nerve nuclei, cerebellum and anterior

horn cells in the spinal cord may also undergo necrosis.

� Cerebral cortical e deep nuclear injury: occurs primarily

with moderate to severe insult that evolves in a gradual

manner. Based on MRI studies, approximately 35e85% of

asphyxiated term infants exhibit predominantly cerebral-

deep nuclear neuronal involvement. Amongst the neu-

rons of cerebral cortex, those in the parasagittal areas of

perirolandic cortex are most likely to be affected. The other

most common neuronal lesion affects putamen in basal

ganglia and thalamus.

Neuroprotective and neurorestorative strategies

C Anti excitatory or

anticonvulsant

� Hypothermia

� Xenon

� Magnesium

� Topiramate

� Lamotrigine

� Phenobarbitone

� Bumetanide

C Anti inflammatory or

anti oxidant

� Hypothermia

� Minocycline

� Indomethacine

� Melatonin

� N-acetyl cysteine

� Allopurinol

� Sodium chromoglycate

C Growth factors

� Nerve growth factor

� Insulin like growth factor

� Brain derived neutrophilic

factor

C Antiapoptotic

� Hypothermia

� Erythropoietin

� Caspase inhibitor

C Multiple mechanisms

� Hypothermia

� Erythropoietin

C Neurorestorative

� Erythropoietin

� Cord blood and

mesenchymal stem cells

Table 2



� Deep nuclear e brain stem injury: occurs primarily with

severe relatively abrupt insult. This is a predominant

lesion in approximately 15e20% of infants with HI insult

with involvement of basal ganglia, thalamus and

tegmentum of brain stem. (Table 1)

Neuroprotective strategies

The insight into biochemical and cellular mechanisms of injury

following HI helps to provide intervention during the therapeutic

window i.e. the time interval after HI during which interventions

may be efficacious in reducing the severity of brain injury. The

evidence of an ongoing neurotoxic cascade and secondary energy

failure following hypoxic ischaemic damage leads to the possi-

bility of neuroprotective strategies being employed in the hours

following perinatal asphyxia to prevent long-term neuro-

developmental sequelae.

Moderate hypothermia initiated within 6 hours of HI insult

improves survival without cerebral palsy or other disability by

about 40% and reduces death or neurological disability by nearly

30%. In view of the definite but incomplete success of hypo-

thermia after HI in reducing disability from HIE, the search is

now on for treatments that might augment neuroprotection and

neurorestoration (Table 2).

Summary

The development of brain injury after perinatal HI insult is an

evolving process initiated during the acute insult and extending

into a reperfusion phase which may last for several days. The

existence of a brief period of normal cerebral metabolism in the

evolution of HIE supports the concept of therapeutic in-

terventions which might ameliorate the later secondary energy

decline and the neurodevelopmental disability associated with it.

Hypothermia and other neuroprotective and neurorestorative

strategies may improve the neurological outcome in HIE. A

FURTHER READING

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lesions in term infants with neonatal encephalopathy. Lancet 2003;

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repairing processes of cerebral damage in perinatal hypoxic-ischemic

encephalopathy. Ital J Pediatr 2010; 36: 63.

Erecinska M, Cherian S, Silver IA. Energy metabolism in mammalian brain

development. Prog Neurobiol 2004; 73: 397e445.

Fatemi A, Wilson MA, Johnston MV. Hypoxic-ischemic encephalopathy in

the term infant. Clin Perinatol 2009; 36: 835e58.



http://refhub.elsevier.com/S1751-7222(14)00028-6/sref1














Practice points

C Hypoxic ischaemic encephalopathy is an important cause of

neonatal mortality and neurodevelopmental morbidity.

C Hypoxic ischaemic insult is a complex injury characterised by

biphasic depletion in high energy phosphate: primary and sec-

ondary energy failure.

C The secondary energy failure has been shown to be related to

long-term neurological outcome.

C Brain injury following hypoxia-ischaemia evolves over hours to

days providing a window of opportunity for intervention.

C Therapeutic hypothermia improves neurodevelopmental outcome

in neonates with moderate and severe hypoxic ischaemic en-

cephalopathy if initiated within 6 hours.


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