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Neonatal hypoxic ischemic encephalopathy-related biomarkers in serum and
cerebrospinal fluid
Hongyan Lv, Qiuli Wang, Sujing Wu, Lihong Yang, Pengshun Ren, Yihui Yang, Jinsheng Gao, Lianxiang Li
PII:
DOI:
Reference:

S0009-8981(15)00401-5
doi: 10.1016/j.cca.2015.08.021
CCA 14083

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Clinica Chimica Acta

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Accepted date:

27 May 2015
22 August 2015
25 August 2015

Please cite this article as: Lv Hongyan, Wang Qiuli, Wu Sujing, Yang Lihong,
Ren Pengshun, Yang Yihui, Gao Jinsheng, Li Lianxiang, Neonatal hypoxic ischemic
encephalopathy-related biomarkers in serum and cerebrospinal uid, Clinica Chimica Acta
(2015), doi: 10.1016/j.cca.2015.08.021

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Neonatal hypoxic ischemic encephalopathy-related biomarkers in serum and
cerebrospinal fluid
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, Lihong Yang , Pengshun Ren , Yihui Yang ,

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Jinsheng Gao , Lianxiang Li

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, Qiuli Wang , Sujing Wu


2,3

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12

Hongyan Lv

Department of Neonatology, Handan Maternal and Child Care Centers, Handan


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056002,Hebei Province, P.R. China; Department of Neonatal pathology, Handan Maternal


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and Child Care Centers, Handan 056002, Hebei Province, P.R. China; Department of Neural

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development and neural pathology, Hebei University of Engineering School of Medicine,


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Handan 056029, Hebei Province, P.R. China; Department of Pathology, Hebei University of

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Engineering School of Medicine ,Handan 056029, Hebei Province, P.R. China

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Corresponding author: LianXiang Li, Professor, Department of Neural development and


neural pathology, Hebei University of Engineering School of Medicine; Department of Neonatal

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pathology, Handan Maternal and Child Care Centers, Congtai Road No. 83, Handan 056029,
Hebei Province, P.R. China
Email: lianxianglm68@163.com
Tel: + 86 3106038483; + 86 3102116095

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ABSTRACT
Neonatal hypoxic ischemic encephalopathy (HIE) is a common disease caused by perinatal

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asphyxia, a major cause of neonatal death, neurological behavior, and long-term disability.

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Currently, the diagnosis and prognosis of neonatal HIE is based on nervous system clinical
manifestations, imaging and electrophysiological examination. These take time and late
diagnosis allows brain injury to occur in newborns, so that infants of many brain injury missed

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the best treatment time , left with varying degrees of neurological sequelae. The use of

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biomarkers to monitor brain injury and evaluate neuroprotective effects might allow the early
intervention and treatment of neonatal HIE to reduce mortality rates. This study reviewed the

mechanism of neonatal hypoxic ischemic encephalopathy in relation to numerous

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brain-related biomarkers including NSE, S-100, GFAP, UCH-L1, Tau protein, miRNA, LDH,

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and CK-BB. In early diagnosis of neonatal HIE, S-100 and activin A seems to be better
biomarkers. Biomarkers with the greatest potential to predict long-term neurologic handicap of

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neonates with HIE are GFAP and UCH-L1 and when combined with other markers or brain
imaging can increase the detection rate of HIE. Tau protein is a unique biological component of
nervous tissues, and might have value for neonatal HIE diagnosis. Combination of more than
two biological markers should be a future research direction.
Keywords: Newborn; Hypoxic ischemic encephalopathy; Biomarker; Serum; Cerebrospinal
fluid

Abbreviations: HIE, Neonatal hypoxic ischemic encephalopathy; MIR, magnetic resonance


imaging; CSF, Cerebrospinal fluid; ATP, Adenosine triphosphate; NSE, Neuron specific

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enolase; MBP, Myelin basic protein; GFAP, Glial fibrillary acidic protein; UCH-L1, Ubiquitin
carboxyl-terminal hydrolase; BDNF, Brain-derived neurotrophic factor; miRNA, MicroRNA;

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MMP-9, Matrix metalloproteinase-9; ICAM-1, intercellular adhesion molecular-1; sLCAM-1,

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Soluble intercellular adhesion molecule 1; VEGF, Vascular endothelial growth factor; SOD,
Superoxide dismutase; MDA, Malondialdehyde; Hs-CRP, High-sensitivity C-reactive protein; IL,
interleukin; TNF-, Tumor necrosis factor-; LDH, Lactic dehydrogenase; CK-BB, Creatine

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kinase BB.

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1. Introduction
Neonatal hypoxic ischemic encephalopathy (HIE) is a neonatal brain injury caused by

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perinatal asphyxia and is a major cause of neonatal death, cerebral palsy and mental

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retardation[1,2]. According to estimates, of the approximately 130 million births worldwide


each year, four million infants will suffer from birth asphyxia, and of these, one million will die
and

similar

number

will

develop

serious

and

long-term

sequelae

including

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neurodevelopmental disorders[3]. In China, the incidence rate of neonatal asphyxia is

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1.1411.7%, and the incidence of HIE in full-term live birth infants is 12/1000 of affected
newborns. Approximately 1520% of affected newborns will succumb within the neonatal

period, and an additional 2530% will develop severe and permanent neurological

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handicaps[4], including cerebral palsy, seizures, visual defects, mental retardation, cognitive

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impairment and epilepsy[5]. Serious harm to a childs physical and mental health causes great
mental and economic burden to the family and society. Currently, the early diagnosis of

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neonatal HIE in the clinic depends on observing clinical symptoms and signs using a
combination of computer tomography (CT), magnetic resonance imaging (MRI), ultrasound
and electroencephalogram (EEG). However, these examinations have different limitations and
effectiveness. Biomarkers in the blood circulation are bio-chemical factors released by specific
tissues or organs, and their expression levels reflect a specific physiological or pathological
state of tissues and organs. The accurate detection of body fluid biomarkers in neonatal HIE is
important and will allow early interventions to reduce neonatal mortality, morbidity and degree
of disability. In addition, biomarker evaluation will be useful for the evaluation of neonatal HIE
therapeutic measures such as mild hypothermia therapy, stem cell activity factor, neural

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nutrition factor, and neuroprotective drugs. With the rapid development of biomedicine,
biomarkers associated with neonatal HIE have been reported; however, there has been

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comparatively less research in neonatal HIE than in adult HIE and the data has not been

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assessed together. Therefore, this study retrospectively summarized neonatal HIE research
related to biomarkers to provide important information to help doctors understand, verify and
apply these biomarkers to gradually establish an efficient, accurate and convenient biomarker

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for the diagnosis and evaluation of neonatal HIE.

2. Pathogenetic mechanisms of HIE

Brain oxygen consumption accounts for 2025% of the human body and is very sensitive

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to hypoxia. Neonatal HIE cerebral injury develops in two phases: The first or primary insult

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dominated the brain tissue energy metabolism disorder and the second orreperfusion phase
dominated the histopathological changes of ischemia/reperfusion. The mechanism of neonatal
free radicals,

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HIE brain injury is not completely understood, it might be related to formation of

effect of lipid peroxidation, intervention of inflammatory factors, effect of excitatory amino acid
toxicity, water channel proteins out of control, abnormal calcium ion channels and neuronal
apoptosis. They affect each other and reinforce each other, forming a multiple cascade chain,
eventually leading to neuronal apoptosis or death, nerve fiber degeneration and disintegration
of the brain tissue injury[6-10]. Histopathological studies have identified characteristic
neonatal HIE brain pathological features including nerve cell degeneration and necrosis,
periventricular leukomalacia, cerebral edema, cerebral infarction, periventricular cyst like
changes, intracranial hemorrhage and cerebellar injury. In the pathological process of cerebral

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hypoxia ischemia, various products produced by brain tissues enter the cerebrospinal fluid and
might be used as blood biomarkers; therefore, monitoring these biomarkers or products might

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help the clinical understanding of HIE.

3. The necessity of testing the biological markers in neonatal HIE


The clinical diagnosis of neonatal HIE and disease severity assessment mainly rely on the

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Sarnat score, brain CT scans, MRI, ultrasound diagnosis and EEG detection methods.

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Because of the influence of the progressive disease process and other factors, the Sarnat
score is subjective, and other tests have certain limitations and effectiveness. After neonatal

HIE onset, there is a time difference in the range of 24 hours between biochemical metabolism

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changes, tissue morphological changes and pathological changes in the brain. Neuroimaging

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studies suggest the appearance of nervous system damage can take up to 72 hours[11].
Therefore, the clinical diagnosis of HIE by CT detection is often greater than 72 hours after

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neonatal HIE insult. Although MRI can observe brain pathological changes hours after
neonatal brain injury, but the early minor brain injury and its injury range is limited, and
neonatal disease is dying at this time, imaging detection has certain difficulties. Amplitude
integrated EEG can detect early changes associated with brain injury, however, interference
from hypothermic environments can reduce the prediction of HIE prognosis and it cannot
determine the time of injury[12]. Therefore, the early monitoring of serum or cerebrospinal fluid
of neonatal HIE related biomarkers is particularly important. A biomarker is the product of
specific tissues and organs, and after the onset (minutes or hours) of neonatal HIE, damaged
brain tissues release specific tissue components or products into the blood or cerebrospinal

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fluid; So , neonatal HIE biomarker expression in blood or cerebrospinal fluid might indicate
brain injury or reflect the extent of damage. Thus, the early clinical detection of blood or

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cerebrospinal fluid biomarkers might allow an earlier diagnosis compared with MIR or CT

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results. This would allow the earlier initiation of intervention measures to improve neonatal
survival and reduce the degree of brain injury. In summary, biomarkers will be an important
basis for the diagnosis and differential diagnosis of neonatal HIE, evaluating the intervention

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and drug efficacy, as well as assessing the severity of illness and determining prognosis. Thus,

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biomarkers can be used to characterize the degree of brain damage, the evolution of disease,

recovery effects and when to end treatment.

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4. Neonatal HIErelated biomarkers

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Clinically used biomarkers include creatine kinase brain isoenzyme (CK-BB), myocardial
enzyme (ME), troponin and others, which aid the diagnosis of neonatal HIE. This article will

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review biomarkers associated with nerve tissues, blood vessels, blood brain barrier, oxidative
stress, inflammation, and metabolic related processes to form a foundation for further study
[Table 1].

4.1 Nerve tissue injury-related biomarkers


Nervous tissue is composed of neurons and glial cells. According to their morphology and
function, glial cells are divided into small glial cells, oligodendrocytes, and astrocytes. Because
these cells have different compositions, metabolites and functions, when subjected to
hypoxic-ischemic injury, the different factors secreted from these cells into blood or

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cerebrospinal fluid can be used to determine the type of nerve cell damage and the extent of
damage [Table 2].

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4.1.1 Neuron specific enolase (NSE)

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NSE is a dimeric isozyme of the glycolytic enzyme enolase, is found in the cytoplasm of
cell with neuroendocrine differentiation and neurons of brain tissue.In addition, red blood cells,
liver, smooth muscle and lymphocytes also express NSE. The content of NSE in blood cells is

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at least 30 times lower than that in brain cells. to detect the level of NSE accurately, serum

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specimens must be prevented from hemolysis. Cerebrospinal fluid and serum NSE levels can
be estimated indirectly to determine the degree of neuron damage and the prognosis of

neonatal HIE. Celtik et al., studied 43 cases of full-term neonates with HIE caused by asphyxia,

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and found that the serum NSE levels of HIE cases was significantly higher than that of the

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control group and the healthy group. ROC curve analysis showed that serum NSE
concentrations greater than 40 mcg/L at 4 and 48 hours after birth could distinguish between

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newborns with no or mild HIE and those with moderate or severe HIE. In addition, a serum
NSE cut-off concentration of 45.4 mcg/L could distinguish between normal infants and those
with a poor prognosis [13]. It was also reported that serum NSE concentrations of HIE patients
with a poor prognosis were 18.08 3.97 ng/ml by radioimmunoassay [14]. In most studies, the
NSE concentration in serum and cerebrospinal fluid was consistent with the severity of the
disease, i.e. the higher the concentration of NSE, the more serious the damage was to
neurons[15-17]. Hypothermia treatment of neonatal HIE cases shows abnormal changes in
blood serum NSE and brain injury neural imaging shows that NSE has important clinical value
for the diagnosis and prognosis of HIE [18]. However, Nagdyman et al. suggested there was

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no difference in serum NSE concentrations in neonates with no or mild HIE and those with
moderate or severe HIE [27]. Therefore, further research and discussion regarding the use of

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NSE for the assessment of neonatal HIE is required.

4.1.2 Myelin basic protein (MBP)

MBP is the major protein component of the myelin sheath, and has a crucial role in the

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maintenance of the myelin structure and function [19]. Under normal circumstances, MBP can

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easily pass through the blood-brain barrier into the cerebrospinal fluid, but only a small amount
is released into the blood stream. In a variety of brain injury or diseases involving white matter

(myelin sheath) damage, the concentration of MBP in the blood or cerebrospinal fluid

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increased rapidly, reflecting the severity of myelin damage. Therefore, MBP can be used as a

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specific biochemical marker of nerve fibers in the brain, which changes due to white matter
lesions or nerve fiber demyelination [20-22]. Clinical studies showed that the levels of serum

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MBP in neonates with moderate or severe HIE was significantly higher than in those with mild
HIE and control group infants [23,24]. However, the few studies of MBP in neonatal HIE have
used a small sample size, therefore, the use of MBP as a biomarker for neonatal HIE requires
further study.

4.1.3 S-100
Protein S-100 is an acidic calcium-bingding proteinin in nervous tissues. It is now known
there are 25 members of the protein S-100 family, among which, S-100A and S-100 are
brain specific and the most important and most active members of the central nervous

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system.Proteins S-100 are synthesized and secreted by astrocytes and Schwann cell.
Under normal circumstances, there are small amounts of protein S-100 in the

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cerebrospinal fluid, blood and urine; however, when the brain tissue is damaged, astrocytes

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release large amounts of protein S100 into the blood and cerebrospinal fluid. Protein
S-100 is a specific indicator to determine and evaluate minor brain damage of neonatal
HIE. Qian et al. demonstrated that a cord blood concentration of S-100 greater than 2.02

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g/L, with a sensitivity of 86.7% and specificity of 88% predicted moderate and severe

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HIE[25]. Because protein S-100 are secreted by astrocytes, the detection of serum
concentrations protein S-100 can also be used to determine blood brain barrier damage

[26] or central nervous system inflammation. The protein S-100 is stable in blood and is

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not affected by hemolysis. It is currently considered a potential biomarker for detecting

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neonatal HIE. Animal experiments showed that the levels of serum protein S100 gradually
increased at 0.51 hours after hypoxic ischemic brain damage, and began to decrease at

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48 hours. Hypoxia/asphyxia is a risk factor for S-100 release, and it is a direct marker of
neuronal brain damage when elevated concentrations of S-100 are detected in plasma or
cerebrospinal fluid. Higher concentration of S-100 are observed in umbilical cord blood
from perinatal asphyxia infants and neonatal HIE patients, serum concentration S-100 of
8.5 g/L at 2 hours after birth could predict the occurrence of severe neonatal HIE [27].
There is also a close relationship between serum concentration of protein S-100 and
clinical grading, because the concentration gradually increases with the severity of disease,
and during random spontaneous recovery or after the initiation of neural protection it
gradually decreases [28,29]. It was recently reported that protein S-100 can be detected in

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saliva to predict neural function abnormalities caused by neonatal asphyxia. The study
showed that asphyxia neonatal neurological prognosis was poor in children when S-100

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concentrations in saliva were significantly higher than in the control group. A cut-off

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value >3.25MoM S-100 achieved a sensitivity of 100% (confidence interval [CI]5-95%:


89.3100.0%), and specificity of 100% (CI5-95%: 98.6100%) for predicted neonatal asphyxia

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for neonatal HIE diagnosis and prognosis.

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leading to abnormal neurological outcomes [30]. Thus far, S-100 is a promising biomarker

4.1.4 Glial fibrillar acidic protein (GFAP

GFAP is a skeletal intermediate filament protein in astrocytes and is symbol of the

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physiological and pathological state of astrocytes. Astrocytes produce GFAP, S-100, matrix

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metalloproteinase (MMP)-9, and other neurotrophic factors. When brain injury occurs, the
serum concentration GFAP is increased. Recent studies showed that GFAP was an ideal

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marker for the detection of ischemic brain damage and astrocyte activity in neonates with
hypoxic ischemic brain damage[31]. In the central nervous system, astrocytes are present
between neurons. The end of neurites are swollen and are attached to the capillary wall
adjacent to or attached to the brain and spinal cord, which participate in the blood-brain barrier
and blood-cerebrospinal fluid barrier. Therefore, changes in serum concentrations GFAP
reflect both changes in astrocytes in the cerebral tissue but also blood brain barrier damage. A
study of hypoxia ischemia in newborn piglets showed periventricular white matter astrocytes
were reduced in number, with a smaller cell size, and decreased GFAP content[32],
suggesting hypoxic ischemia may cause astrocyte damage and increased cell death. The

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increase of serum GFAP levels was closely related to the severity of HIE. If a higher serum
GFAP concentration was sustained at birth, it often indicated moderate or severe neonatal HIE.

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When serum GFAP concentrations were >0.08 pg/ml, the positive predictive value was 100%

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for predicting infants with abnormal outcomes [33]. Reports also showed that at a cut-off value
of 0.07 ng/ml, the sensitivity and specificity of GFAP for the diagnosis of neonatal HIE was
77% and 78%, respectively [41]. In clinical practice, changes in serum GFAP concentrations

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from birth to 96 h are especially concerning, as it has important value to predict later motor

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development as well as basal ganglia and cerebral white matter damage. In these cases,
physicians or doctors should not hesitate to initiate early hypothermia therapy or nerve

protective measures. To reduce or mitigate the occurrence of neurological sequelae, Ennen et

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al. detected serum GFAP concentration in HIE neonates, HIE neonates with MRI

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abnormalities, and healthy newborns within 1 week after birth, they found that serum GFAP
concentrations in neonates with HIE were higher than in healthy newborns and that serum

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GFAP concentrations in HIE neonates with MRI abnormalities were higher than in neonates
with HIE (i.e. by normal imaging). This suggested that serum GFAP concentrations increased
in the first week after birth can be used to assess the severity of brain damage and can screen
the treatment of neonates [34]. From these research results, GFAP appears to be a promising
biomarker for the diagnosis and prognosis of neonatal HIE.

4.1.5 Ubiquitin Carboxyl-terminal hydrolase L1 (UCH-L1)


UCH-L1 is a protein in the cytoplasm of neurons, also known as protein gene product 9.5. It
is widely distributed in the vertebrate brain and in human neuroendocrine cells, and is an

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important member of the ubiquitin proteasome system, accounting for about 2% of all brain
proteins. Because of its high and specific expression in brain tissue, elevated level of serum

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UCH-L1 have been suggested to be a marker of nervous system injury[35-37] such as acute

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cerebral ischemic disease[38] and early stage of severe traumatic brain injury [39] . Recent
studies reported high levels of UCH-L1 in the umbilical cord of neonates with HIE, related to
cerebral cortex injury and subsequent movement and cognitive development. Receiver

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operating characteristic (ROC) curves showed that serum UCH-L1 concentrations of ~131

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ng/ml, had a specificity of 100% for the diagnosis of neonatal HIE, whereas a concentration of
~28 ng/ml had a specificity of 95%[40]. Another ROC curve analysis using UCH-L1 for the

prognosis of children with neurological abnormalities after 15-18 months of postnatal life

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showed the area under curve (AUC) was 0.703 [95%CI: 0.6870.742] [41]. Especially, the

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mean serum UCH-L1 concentration at 24 hours was 8.62 ng/ml for subjects with a poor
development outcome and 2.05 ng/ml for subjects with a good prognosis[42]. To date, the

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research of UCH-L1 is still less for the diagnosis and assessment of neonatal HIE as well as
the effect of long-term nerve development in neonatal HIE, Therefore, further research and
development is required.

4.1.6 Brainderived neurotrophic factor (BDNF)


BDNF is widely distributed in the central nervous system and is secreted by central
nervous system neurons and astrocytes to promote the growth, differentiation, regeneration,
and repair of neurons. Although BDNF is a marker of brain nutrition factors and central
nervous system damage, there have been few studies investigating the relationship between

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BDNF and neonatal HIE. Cord blood BDNF concentrations of premature infants with severe
cerebral hemorrhage were significantly lower than in healthy controls[43]. However, the serum

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BDNF concentrations in patients with severe asphyxia leading to encephalopathy were

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significantly higher than in the control group, especially within 72 hours of postnatal life. If the
serum BDNF concentrations of neonates with HIE are persistently elevated, this suggests

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severe brain injury and a poor prognosis[44].

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4.1.7 Tau protein

Tau protein is a neuronal scaffolding protein that participates in actin filaments composed

of neurons. When neurons are damaged neurons tau protein is released. Therefore, the

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degree of neuronal damage is reflected by the detection of serum or cerebrospinal fluid levels

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of Tau protein. Increased tau protein in adult ischemia or animal experiments of traumatic
brain injury suggested it might be a sensitive indicator of early cerebral ischemia[45]. Serum

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Tau protein concentrations were significantly increased in bilirubin encephalopathy of


newborns, and there was significant correlation between the degree of brain injury severity
and concentration of Tau protein[46]. These studies suggest that a close relationship between
tau protein and brain injury exists. Unfortunately, there has been no research on Tau protein
and neonatal HIE; therefore, further research and development is needed.

4.1.8 MicroRNA (miRNA)


miRNA belongs to a class of single molecules of RNA containing 19-23 non coding
nucleotides, in multicellular animals and plants. miRNA plays a role in regulating gene

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expression by binding to the target gene mRNA at post transcription. In recent years, miRNA
has been shown to be involved in the pathophysiology of hypoxic ischemic encephalopathy,

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including the regulation of excitatory amino acid toxicity, oxidative stress, inflammatory

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reactions, and apoptosis. Animal experiments investigated the miRNA expression profile of the
cerebral cortex in neonatal HIE brain injury rats using microarray technology. It was found that
5 miRNAs were up-regulated and 29 miRNAs were down regulated[47]. In the small RNA

closely

related

to

the

occurrence

and

development

of

hypoxic

ischemic

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were

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family, miRNA-199a is known as a specific marker of neural tissue[48]. miRNA in brain tissues

encephalopathy[49,50]. Recently, miRNA-21 expression was shown in astrocytes in brain

tissues[51]. The study of 49 cases of neonates with HIE showed the serum miRNA and

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hypoxia induced factor 1a mRNA concentration was significantly higher than in the healthy

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control group, and increased concentrations of serum miRNA-21 indicated neonatal HIE [52].
Therefore, miRNA-21 might be a marker for the early diagnosis of neonatal HIE, although

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further experiments and clinical verification are needed.

4.1.9 Activin A

Activin A is member of the transforming growth factor (TGF-) superfamily. It is a trophic


factor that regulates neuron proliferation and differentiation. In the central nervous system, the
activin A receptor is highly expressed in neurons, and is upregulated when neurons are
activated[53]. Animal experiments showed that activin A has a protective effect on brain injury
such as cerebral hypoxia ischemia[54] especially within 1 hour of postnatal preterm infants.
Increased serum activin A indicates intraventricular hemorrhage[55]. Measurement of

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cerebrospinal fluid activin A in full-term asphyxia neonatal infants indicated it was higher in
moderate or severe neonate HIE compared with no or mild HIE. The cerebrospinal fluid activin

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A concentration was > 1.3 g/L and the probability of the asphyxia neonate developing

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neonatal HIE (positive predictive value) was as high as 100%. The sensitivity and specificity
for the diagnosis of neonatal HIE was 100% [95%CI: 69.0100%] and 100% [95%CI:
94.3100%][56]. Because urine samples were obtained for convenience, the same

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researchers tested urine activin A in 10 of 12 moderate or severe neonates with HIE. A urine

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activin A concentration of >0.08 g/L was observed in moderate or severe neonates with HIE
and was significantly higher than in neonates with no or mild HIE[57]. Therefore, cerebrospinal

fluid activin A concentrations after neonatal asphyxia might be a reliable early diagnosis

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indicator for the prediction of neonatal asphyxia and neonatal HIE.

4.2 Brain vascular and blood brain barrier-related markers

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4.2.1 Matrix metalloproteinase-9 (MMP-9)


In addition to S-100, GFAP can be used as a marker of the blood brain barrier. MMP-9 can
also assume this role, which involves the degradation of brain vascular basement membrane
components such as collagen IV, laminin and fibronectin. Under the effect of oxygen free
radicals and other inflammatory mediators, MMP-9 is activated, the basement membrane of
the blood brain barrier is damaged, increasing permeability and causing secondary vascular
source cerebral edema. Many studies have shown that MMP-9 is involved in the
pathophysiology of neonatal HIE [Table 3]. Measurement of serum MMP-9 concentrations of
94 neonates cases with mild, moderate or severe HIE showed that the serum MMP-9

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concentrations of all HIE group patients were significantly higher than those in control
group[58]. Serum MMP-9 concentrations are also related to the time of HIE onset, as serum

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MMP-9 concentrations in neonates with HIE were gradually increased within 1-3 days after

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HIE insult, and showed a downward trend on day 7[59], indicating that inflammatory mediators
were involved in the pathological process after neonatal HIE onset. Serum MMP-9 levels were
related to the severity of neonatal HIE, and were significantly higher in neonates with severe

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HIE than in neonates with mild and moderate HIE[60]. A sustained increase of serum MMP-9

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concentrations in neonates with HIE aggravates blood brain barrier damage, causing
secondary brain edema, further aggravating brain tissue damage. Therefore, clinical trials of

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MMM-9 antagonists to block or reduce secondary brain edema and brain damage are needed.

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4.2.2 Vascular endothelial growth factor (VEGF)


VEGF is an angiogenic factor secreted by astrocytes and microglia cell. VEGF and its

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receptor is overexpressed in hypoxia ischemia[62]. VEGF has protective effects on neurons


and glial cells, and can promote the proliferation and angiogenesis of vascular endothelial cells.
VEGF protects cerebral cortex neurons from hypoxic injury, promotes the proliferation of
neuron precursor cells, and survival, repair and regeneration of neurons in the cerebral cortex
after hypoxia ischemia[61]. Animal experiments showed that during brain hypoxia ischemia,
brain tissue VEGF mRNA expression was enhanced and reached a peak at 12 hours of
postnatal life, for a duration of 14 days or even longer[62]. When VEGF was administered to
neonatal HIE rats, brain tissue damage was reduced, and the phosphorylation of protein
kinase B and extracellular signal regulated kinase 1/2 was increased in the cerebral cortex[63],

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suggesting that VEGF has a protective effect on brain tissues. The relationship between VEGF
and neonatal HIE is rarely reported. Tan et al., demonstrated that cerebrospinal fluid and

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plasma VEGF concentrations were significantly higher in 38 cases of neonates with HIE than

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in controls. Furthermore, the cerebrospinal fluid and plasma VEGF concentrations were
increased with increased severity of neonatal HIE [64]. This conclusion was later confirmed by
another study[65]. Currently, it is thought that VEGF is involved in the pathophysiology of brain

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injury after neonatal asphyxia. However, cerebral blood vessels are just one part of the

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systemic vascular system and the serum VEGF concentrations of neonates with asphyxia

might be affected by VEGF secreted from non-brain tissues.

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4.3 Oxidative stress-related markers

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4.3.1 Superoxide dismutase (SOD) and Malondialdehyde (MDA)


Free radicals are a class of harmful substances produced from Free radicals was a class of

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harmful substance from the metabolic disorders, and are important factor that cause brain
damage after cerebral hypoxia ischemia regardless of the type of brain injury. Most free
radicals cause lipid peroxidation of the cell membrane, SOD is an antioxidant enzymes that
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removes excess oxygen free radicals (O and OH ) to protect cells from free radical damage,
Its activity level reflects the oxygen free radical scavenging ability of the body. Because the
brain contains a large amount of unsaturated fatty acids, It is vulnerable to the attack of oxygen
free radical, react to lipid peroxidation and malondialdehyde formation . Therefore, the level of
MDA content reflects the extent of oxidative damage to cells [Table 4]. The change of SOD
activity and the content of MDA indicate brain injury to a certain extent. Free radicals might be

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involved in the pathophysiology of HIE. Excess free radicals consume a large amount of SOD,
produce a large amount of MDA, promote the release of inflammatory factors in brain tissues,

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nerve cell apoptosis, and increased permeability of the blood brain barrier in neonatal HIE[66].

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A study of 50 cases of asphyxiated full term newborns found serious asphyxia resulting in the
death of neonates with HIE, the concentrations of MDA and glutathione peroxidase in plasma
and cerebrospinal fluid were significantly higher than infants who survived [67]. In neonatal

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asphyxia with epilepsy, serum MDA concentrations were significantly higher than those without

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seizures[68]. Moreover, in the development of neonatal HIE, serum MDA concentrations were
increased with the degree of disease, which was confirmed by imaging analysis[69]. Clinical

studies found that in the acute stage of HIE, serum SOD concentrations were significantly

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decreased compared with a healthy control group, and MDA concentrations were significantly

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increased[70,71] after the onset of ischemia hypoxia. Presumably a large amount of


superoxide anions were produced after brain tissue injury and a large amount of SOD was

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consumed. Therefore, serum SOD and MDA concentrations can be used for the early
prediction of neonatal HIE[72]. However, asphyxia leading to neonatal HIE affects the brain
and other tissues and organs, which also produced many free radicals,the detection of serum
SOD and MDA concentrations alone lacks specificity.
4.4 Inflammationrelated markers
In flammatory responses are an important link in neuron apoptosis and death in neonanatal
HIE. Many types of immune cells and inflammatory factors are involved in the occurrence,
development and recovery of brain injury[73]. Neonatal hypoxia ischemia can promote brain
tissues to produce inflammatory factors, further aggravating brain tissue damage [Table 5].

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4.4.1 High-sensitivity C-reactive protein (Hs-CRP)
Hs-CRP is member of the pentraxin family and is a sensitive marker of inflammatory

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reaction and tissue injury. The concentration of Hs-CRP increased rapidly in brain tissues after

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hypoxia ischemia. Shang et al. studied 74 cases of neonates with HIE and showed that serum
Hs-CRP, interleukin (IL)-6, and tumor necrosis factor (TNF)- concentrations were significantly
higher than in the control group, and that levels were in severe HIE were significantly higher

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than in mild HIE. It was also found that the serum IL-6, TNF-, and Hs-CRP concentrations

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were significantly higher in those with a poor prognosis compared with those with a good
prognosis[74], indicating that these markers were closely related to the severity and prognosis

of HIE. It was reported that the serum Hs-CRP concentration of neonates with HIE reached a

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peak at day 3 of postnatal life, and then began to decrease[75]. When the serum Hs-CRP

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concentration of neonates with HIE cannot be reduced, the prognosis is often poor. At this
point, anti-inflammatory treatments can prevent excessive inflammation in the brain and tissue

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damage. To reduce the interference of inflammatory factors in tissues outside the brain after
neonatal asphyxia onset, we should actively explore the changes of cerebrospinal fluid
Hs-CRP concentrations of neonatal HIE, to determine its sensitivity and specificity.
4.4.2 Interleukins
Interleukins are a large family of cytokines. Many studies have reported links between
interleukins and neonatal HIE pathophysiology and repair[76].
IL-1 promotes brain damage in the central nervous system. The mechanism of brain
damage induced by IL-1 involves the release of free radicals, stimulating inflammatory
reactions and enhancing the toxicity of excitatory amino acids. Relevant data show that cord

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blood and peripheral venous blood of IL-1, IL-8, and TNF- concentrations in neonatal HIE
patients were higher than in the control group, and that serum IL-1 concentrations and

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neonatal HIE scores were positively correlated. Elevated levels of serum IL-1 can predict

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neurological abnormalities in neonatal HIE patients after 6-12 months of postnatal life[77]. Aly
et al., studied 24 cases of neonates with HIE and found that blood and cerebrospinal fluid
IL-1 , IL-6, and TNF- concentrations were significantly higher than in the control group,

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demonstrating that cerebrospinal fluid IL-1 was closely related to the diagnosis of neonatal

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HIE[78].Therefore, the detection of cerebrospinal fluid IL-1 concentrations can help


determine the prognosis of neonatal HIE patients.

IL-6 mainly produced by glial cell inhibits the synthesis of TNF- and IL-1, promotes

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nerve growth factor secretion, and has a protective effect on the central nervous system.

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owever, high concentrations of IL-6 can induce inflammation, increase vascular permeability
and the occurrence of secondary cerebral edema. Chiesa et al., studied the cord blood of 50

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infants with asphyxia and 113 normal full-term neonates and showed that IL-6
concentrations were significantly higher in infants with asphyxia than in healthy full-term
neonates, the results show that serum IL-6 concentrations were considered to be helpful for
the diagnosis of brain injury in neonatal asphyxia[79]. According to the study, IL-6 levels
can also be used to determine secondary cerebral edema and poor prognosis[80], but this
study lacked sensitivity and specificity detection. Therefore, further studies to investigate
IL-6 in serum as a biomarker for the diagnosis of brain injury in neonatal asphyxia are
warranted.
IL-8 is a neutrophil chemotaxis factor that recruits neutrophils to lesions, and through

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enhanced IL-1 and TNF-a neurotoxicity, increases brain tissue injury. Measurement of serum
IL-8 in 32 cases of neonates with HIE showed that in the acute phase of neonates with HIE,

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the concentrations of serum IL-8 were significantly higher than in the control group, and that

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the more severe the HIE, the higher the serum IL-8 level. However, after the treatment and
recovery period, IL-8 decreased, suggesting that IL-8 participated in the neonatal HIE
pathophysiology process[81]. Recently, Youn et al., [82] studied 13 cases of neonatal HIE

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with epilepsy, and showed that most inflammatory factors in the serum were decreased after

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48-72 hours of postnatal life; however, serum IL-8 levels remained high indicating IL-8 might
be an early biomarker for the diagnosis of neonatal HIE with epilepsy.

IL-10 plays a protective role in brain tissue by inhibting the secretion of IL-1, IL-8 and

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TNF-a, inhibiting the production of leukocyte aggregation and chemokines, and reducing

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inflammatory responses in the brain. A clinical report showed that serum IL-10 concentrations
in the acute phase of neonates with HIE were significantly higher than in the control group,

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presumably to reduce the hypoxic strss response[83]. After mild hypothermia treatment,
serum IL-10 concentrations in neonatal asphyxia with brain injury were decreased [84].
IL-18 is an anti-inflammatory factor that stimulates the expression IL-1 and IL-8, Therefore,
IL-18 can protect brain tissues, and can aggravate brain tissue damage[85]. Serum IL-18
levels were shown to reflect the pathological and physiological process of neonatal HIE[86].
Most studies reported that serum IL-18 concentrations of neonates with HIE were higher than
those in control groups and that serum IL-18 concentrations in neonates with moderate and
severe HIE were significantly higher than in neonates with mild HIE, which correlated with the
severity of clinical disease [87]. Other studies reported that in the first days after birth, serum

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IL-18 concentrations of full term infants with HIE and seizures were significantly higher than
those without seizures [88]. However, the relationship between serum IL-18 concentration and

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the development of long-term neurological function in neonatal HIE is unknown.

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4.4.3 TNF-

TNF-a promotes the synthesis and release of IL-1 and IL-8, the induction of apoptosis of
nerve cells, disruption of the blood brain barrier, and aggravates brain damage. Oygr et al.,

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[89] studied 30 cases of term infants with HIE divided into two groups and after 12 months of

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postnatal life showed 11 infants had no abnormal neurological signs (normal group), 14 cases
showed neurological abnormalities (abnormal group) and 5 infants died soon after diagnosis.

Cerebrospinal fluid levels of IL-6 and TNF- were significantly higher in the abnormal group

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compared with the normal group, and IL-6 was a better than TNF- at predicting neonates with

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HIE. Other studies indicated that TNF-a is an inflammatory factor involved in the early onset of
neonatal HIE that peaks at 24 hours after birth, and the TNF- concentration was closely

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related to the severity of HIE and the prognosis of the patients[90-92]. Serum TNF-
represents inflammatory reactions of many tissues and organs after neonatal HIE, whereas
TNF- concentrations in the cerebrospinal fluid are limited to brain tissue; therefore, the
detection of TNF- in the cerebrospinal fluid is more valuable to understand the pathological
process of neonatal HIE and predict brain damage.
4.4.4 Intercellular adhesion molecule-1 (ICAM-1)
ICAM-1 is an important member of the immunoglobulin superfamily that mediates
recognition and binding of cell-cell adhesion, extracellular to cells and between plasma
proteins. ICAM-1 exists as soluble and membrane types. The membrane type is located on the

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cell membrane and soluble type is found in body fluids. There is no qualitative difference in
function between the 2 forms, but the soluble form is used in clinical practice. ICAM-1 induces

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and recruits leukocyte aggregation, migration and adhesion in inflammatory conditions,

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participates in the activation and proliferation and apoptosis of cells[93], cell-cell adhesion,
maintaining of normal tissue structure, regulation of inflammation and immune. Under normal
circumstances, ICAM-1 is expressed at low levels, but when exposed to various stimuli, such

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as inflammatory cytokines IL-1, TNF-, interferon- or endotoxin, its expression was

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increased. After binding of ICAM-1 and its ligand, promote leukocyte adhesion to vascular
endothelial cells, causing a series of cascade reactions, so that the white cell activation with

directional migration and other changes across the endothelium, which cause brain edema

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and nerve cell damage. It was confirmed that ICAM-1 was involved in the pathogenesis of

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neonatal HIE, and aggravated brain damage when ICAM-1 was overexpressed. A study of 45
cases of neonates with HIE showed that serum concentrations of soluble ICAM-1 in mild,

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moderate and severe neonates with HIE were significantly higher than in controls, and were
positively correlated with the clinical grading. The more severe the neonatal HIE, the higher
the serum soluble ICAM-1 concentration[94]. Another study demonstrated similar data[95].
Recent reports showed that serum soluble ICAM-1 concentrations in low birth weight asphyxia
neonatal patients were significantly higher than in the control group, and that serum soluble
ICAM-1 concentrations, Apgar score, umbilical artery blood pH value and lactate values had
significant positive correlation (P<0.05) [96]. Perinatal hypoxia asphyxia is the cause of brain
tissue and vascular endothelial cell injury, and changes in serum ICAM-1 concentrations can
help understand the pathological process of neonatal HIE.

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4.4.5 Selectins
early

stage

of

ischemic

cerebral

vascular

disease,

selectins

the

induce

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In

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leukocyte/endothelial interactions. L-, P- and E-selectin are associated with neutrophil


migration. When endothelial cells were stimulated, P-selectin has an important role in the initial
migration of neutrophils[97]. In comparison with adults, P-selectin is expressed at lower levels

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in activated platelets of newborns[98] and in endothelial cells. Similarly, in full-term infant

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neutrophils, L-selectin expression was significantly lower than in adult stimulated or


unstimulated neutrophil cells[99]. A study of 63 cases of neonates with HIE showed serum

P-selectin concentrations were positively correlated with the degree of neonatal HIE[100,101].

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However, L- selectin did not reflect the existence of asphyxia infants with HIE or the severity of

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neonatal HIE [102]. Although P-selectin might reflect the condition of neonatal HIE, it needs to

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be combined with imaging monitoring, to increase its sensitivity and specificity.

4.5 Metabolism-related markers


Perinatal asphyxia is a major cause of neonatal HIE, caused by brain hypoxia ischemia,
which often leads to increased anaerobic glycolysis, decreased ATP production, the
accumulation of acidic metabolites, increased blood levels of cerebrospinal fluid lactic acid,
pyruvic acid and other substances. Because ATP production is reduced, energy transfer of
neurotransmitter ions such as glutamate and calcium occurs; therefore, clinical testing of
metabolites might also provide relevant information for the diagnosis of neonatal HIE [Table 6].

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4.5.1 Lactic dehydrogenase (LDH)
LDH is a glycolytic enzyme that exists in neuron cytoplasm and mitochondria, and its role is

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to catalyze the oxidation of lactate to pyruvate. At neonatal HIE onset, glycolysis, LDH activity

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and lactate production is increased in the cerebrospinal fluid or blood. LDH is commonly used
for myocardial enzyme detection. However, recent studies showed increased LDH
concentrations in serum of neonatal HIE, but not in cardiomyopathy; therefore, the combined

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detection of LDH and NSE index are used. Results showed that serum LDH and NSE levels of

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neonatal HIE patients were significantly higher than that of normal full-term newborns, and as
there was a significant correlation between serum LDH and NSE concentrations and the

severity of disease [103]. In neonatal HIE patients with a poor prognosis, significantly higher

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levels of serum lactic acid, lactate dehydrogenase, aspartate amino transferase, alanine

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aminotransferase and serum creatine kinase (CK) were observed compared with neonatal HIE
patients with a good prognosis. If LDH analysis is combined with MRI detection, the diagnosis

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rate was greatly improved[104]. The sensitivity and specificity of LDH alone for the diagnosis
of HIE was lower (79% and 56%, respectively) than if serum CK, LDH, uric acid and lactic acid
were

simultaneously

detected

(specificity

and

sensitivity

were

87%

and

94%,

respectively)[105]. Therefore, detection of serum CK, LDH and uric acid might have an
important role in neonatal HIE diagnosis and prognosis evaluation.

4.5.2 Creatine kinase BB isoenzyme (CK-BB)


CK-BB is located in the cytoplasm and organelles of neurons and glia. CK-BB reflects the
extent of neuron and glial cell damage[106]. Early research data showed that increased

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activity of CK-BB in serum within 6-12 hours of postnatal life was associated with neurological
functional deficit in the future[107]. The use of serum CK-BB concentrations alone for the

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diagnosis of neonatal HIE, showed the cut-off value was 1196 IU/L, AUC was 0.78 (95% CI:

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0.65-0.91), and sensitivity and specificity were 90% and 57%, respectively. If several indicator
were combined, the sensitivity and specificity were greatly improved (sensitivity 94%,
specificity 87%) [27,105]. However, follow-up data showed that no patients with neurological

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abnormalities showed elevated levels of serum CK-BB activity after 4 hours of postnatal

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life[108]. It was considered that levels of serum CK-BB were not a good indicator for the
prognosis of neonatal HIE with long-term neurodevelopmental delay[109,110]. However, many

studies report that levels of serum and cerebrospinal fluid CK-BB are positively correlated with

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neonatal HIE [111,112], especially, asphyxia infants at birth. If more than two kinds of

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biomarkers are combined better results can be achieved.


4.5.3 Glutamate

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Glutamate is an excitatory amino acid in the central nervous system that mediates
excitotoxicity, which participates in the pathophysiological processes of brain ischemia. After
neonatal HIE insult, hypoxic ischemia in brain tissues causes metabolic disorders, ATP
synthesis deficiency, inhibition of glutamate transport, and the accumulation of a large amount
of glutamate in the neuronal synaptic gap, resulting in neuronal apoptosis or death. Therefore,
serum or cerebrospinal fluid glutamate concentrations reflect the extracellular glutamate
concentration of brain tissue. Clinical observation showed increased levels of serum glutamate
after neonatal HIE insult[113,114]. Within 24 hours, the increase of glutamate was significant,
and reached a peak at day 3 of postnatal life. At day 7 (recovery period) the levels returned to

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normal, and serum glutamic acid concentrations were closely related to the severity of HIE
[115]. The concentration of glutamate in the cerebrospinal fluid of neonates with severe HIE

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were significantly higher than in those with mild or moderate HIE[116]. Thus, because

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glutamate is a specific product of the brain, it is a sensitive biomarker for brain injury. If
glutamate concentrations can be monitored early, they might aid the early diagnosis of
neonatal HIE.

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5. Ideas for establishing an optimized combination of biomarkers

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Because of the severity of neonatal HIE, patients show different pathological changes in
the brain; thus, the expression of biomarkers also show differences. Furthermore, a unified

standard between the degree of neonatal HIE brain injury and the concentration of relevant

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biomarkers in body fluids had not been established. Although brain-specific biological markers

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can be used, there are numerous heterogenous factors that affect the results of tests.
Therefore, the use of a certain biomarker to detect neonatal HIE is limited. Therefore, two or

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more biomarkers should be combined to establish an optimized scheme. For example, S-100
combined with GAFP, S-100 combined with CK-BB and LDH, GAFP combined with IL-6, or
UCH-L1 combined with Activin A. This method might be useful in determining the development
of HIE and improve the accuracy of diagnosis and prognosis. This might also avoid some of
the non-specific limitations of the biomarkers, resulting in a more comprehensive picture of the
pathological state of neonatal HIE. However, to achieve this goal, many clinical studies are
required to verify and refine the use of biomarkers.
6. Conclusion
HIE causes serious injury to newborns. Clinical imaging is the basic assessment method for

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neonatal severe injury but it has some limitations. Therefore, searching for reliable biomarkers
has become one a research hot spot. Recently, some biomarkers have been found that might

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complement the imaging of abnormalities to aid the prognosis of neonatal HIE. Currently,

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based on the accuracy rate of neonatal HIE assessment; there is no single marker that can
diagnose disease with 100% certainty. In addition to the use of LDH and CK-BB markers,
S-100 and Activin A might be better biomarkers. To predict the long-term neurologic handicap

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for neonates with hypoxic ischemic encephalopathy, GAFP and UCH-L1 are the most useful

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biomarkers, especially if they are combined with other markers or with imaging monitoring. It is
known that neural tissues are the only source of Tau protein, therefore it should be actively

developed for clinical use in the diagnosis and treatment of neonatal HIE. In addition, future

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research should optimize the use of multiple biomarkers to establish a unified, standard

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method to improve the accuracy of neonatal HIE diagnosis.

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Competing interests

The author has no competing interests.


Acknowledgments
The authors sincerely acknowledge the financial assistance provided by the Health and Family
Planning Commission of Hebei, Key project of medical science research of Hebei Province
(NO:2015033)

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infants:relationship

to

hypoxic-ischemic

encephalopathy.

Acta

ACCEPTED MANUSCRIPT
Table 1

Change in

Elevated levels

Serum and

of Pathology

Association Reference

Sensitivity

curve

CSF level

SC
R

Nerve tissue
markers

Released after

Severe brain

death neurons

i njury or poor

andastrocytes

prognosis

Necrosis and

Severe brain

decomposition
of brain white

Released after

TE

S-100

matter

Brain injury

death astrocytes

or poor

but can released

prognosis

References

(%

Severe HIE:

Severe HIE:

79%

70%

NU

MBP

MA

NSE

Specificity

IP

Biomarker

summary of neonatal hypoxic ischemic encephalopathy related biomarkers

[13-17

Poor prognosis Poor prognosis

IC

84 %

70%

UK

UK

Severe HIE:
83%-86.7%

Severe HIE:

[23,24

[25,27-29

88%-95%

CE
P

from other tissue


damage

Released after

Severe brain

death astrocytes

injury or poor

AC

GFAP

Severe HIE:
77%

prognosis

Severe HIE:

31,33,34,41

78%
Prediction
abnormal
outcome:
100%

UCH-L1

DBNF

Released after

Brain injury

central neuron

or poor

injury

prognosis

Secretion neuron

Brain injury

Severe HIE

[40,41]

95%- 100%

IC

UK

UK

UK

UK

UK

UK

[43,44]

PVH) and astrocytes


Tau

UK

Released after

Brain injury

IC

neuron injury

miRNA-21

Released after

Brain injury

IC

52

ACCEPTED MANUSCRIPT
astrocytes injury

Activein A

Released after

Severe HIE

prediction

HIE100%

HIE100%

or other cell

BV and BBB
markers

Inflammatory

inflammation,

reaction,

hypoxia sti-

vascular injury

mulated of

cerebral edema

microglia cells,

in HIE

neuron, macrophage,T lymph-

Released after

UK

UK

[ 58-60

Brain injury,

IC

UK

UK

33,64,65

IC

UK

UK

70-72

IC

UK

UK

68-72

UK

[74,75]

TE

ocytes, etc.

VEGF

IC

NU

Released after

MA

SC
R

IP

injury

MMP-9

56

neuron injury

prediction

astrocytes,

process of

microglia cells

HIE,

AC

markers

pathological

CE
P

Oxidative stress

hypoxia ischemia

SOD

MDA

Released after

Early stage

neuron injury

in HIE

Early stage
in HIE

Inflammation
marker
Hs-CRP

Released after

inflammatory

Severe brain

IC

UK

injury,

cells were
stimulated
IL-1

Released after

Acute phase

astrocytes

HIE, severe

vascular endothelial

brain prognosis

Prediction

Prediction

abnormal

abnormal

neurologic neurologic

77,78

ACCEPTED MANUSCRIPT
cells were stimu-

outcome: outcome:

lated

IL-6

88%

Glial cells

Brain injury,

IC

80%

UK

UK

[33,41,79,80

Glial cells,

Acute phase

activated

in HIE or poor

monocytes /

prognosis

macrophages

Monocyte /

Acute phase

macrophages,

in HIE or poor

B lymphocytes

prognosis

TNF-a

Glial cells,

Severe brain

r elease

injury

Glial cells,
vascular
endothelial

Severe brain

UK

81,82

UK

UK

[83,84

IC

UK

UK

86-88

IC

UK

UK

89-92

UK

UK

[94-96

UK

100,101

HIE

HIE

103-105

diagnosis:

diagnosis:

TE

IL-18

MA

release

UK

IC

NU

release.

IL-10

IC

IP

SC
R

IL-8

release

injury

CE
P

, macrophages
release

Vascular endo-

Acute phase

thelial, white

in HIE or poor

AC

ICAM-1

blood cells,

IC

prognosis

macrophages
release

P- Selectin

Stored in platelet

HIE pathology

IC

UK

a granules, platelet
endothelial cells,
macrophages and
expressed on cell
surface

Metabolic markers
LDH

Released after

Severe brain

neuronal death

injury or poor
prognosis

79%

56%

ACCEPTED MANUSCRIPT

CK-BB Released after


neuron, glial
cells , placenta

Severe brain

injury or poor

HIE 105,107,110-111

HIE
diagnosis:

prognosis

90%

diagnosis:
57%

Severe brain

neuron injury

injury

IC

UK

UK

113-116

SC
R

acid

Released after

IP

Glutamic

lung,iKidney,

vessel;

NU

Note: HIE, neonatal hypoxic ischemic encephalopathy CSF, Cerebrospinal fluid;


BBB, Blood brain barrierPVH, Premature ventricular hemorrhage;

NSE, Neuron

MBP, Myelin basic protein; GFAP, Glial fibrillary acidic protein; UCH-L1,

MA

specific enolase;

BV, Brain

Ubiquitin carboxyl-terminal hydrolase-L1; BDNF, Brain-derived neurotrophic factor;

miRNA,

Superoxide dismutase; MDA,Malondialdehyde; Hs-CRP,High-sensitivity C-rective

TE

SOD,

MicroRNA; MMP-9, Matrix metalloproteinase-9; VEGF, Vascular endothelial growth factor;

CE
P

protein; IL-1, Interleukin-1; IL-6, Interleukin-6; IL-8, Interleukin-8; IL-10, Interleukin-10; IL-18,
Interleukin-18; TNF-a: Tumor necrosis factor-a; ICAM-1: intercellular adhesion molecular-1;

AC

LDH: Lactic dehydngenase; CK-BB, Creatine kinase BB;


Unknown;

Y, Yes;

IC, Incomplete; UK,

, Limited application , General application; , Application is more,

evaluation is more ; , Future research directions

ACCEPTED MANUSCRIPT
Table 2
Summary of clinical studies investigating the nerve injury markers in neonates with HIE
Clinical

marker

reference

grouping

Specimen Assay
(unit)

Detection time
After birth ( h or d)
Mean value

NSE
Liu CY, et al

Serum

[14]

Normal

Mild HIE

12

RID
(ng/ml)

( Range)

(1d)

3.92

(1.47)

NU

[ 2005]

Geometric

SC
R

(SD)

Median

Author and

IP

Biological

8.42

ModerateHIE 14

Severe HIE

MA

(1.84)

ELISA

Normal

25

(ug/L)

Mild HIE

20

Serum

TE

Yang JL, et al.


[15]

CE
P

[1997]

AC

(2.30)

(1d)

(3d)

(7d)

8.0

7.9

7.6

9.6

CSF
Control

21.76

9.0

7.9

(2.7)

(2.6) (1.4)

11.8

10.4

(3.6)

(3.5) (2.3)

8.4

20

Feng X, et al
[16]

(1.12)

(1.5) (1.7) (1.0)

ModerateHIE 20

Severe HIE

11.10

ELISA

1d .

(ug/L)

1.33

[1997]

(0.72)
Mild HIE

1.45
(1.42)

ModerateHIE

4.21
(2.70)

Severe HIE

8.29
(4.75)

Nagdyman N,et al.


[27]

Control

20

[2001]

Cord

ELISA

( 24h

blood

( ug/L )

29.6
(17.8-55.9)

No or Mild HIE 22

48.9
(20.1-74.7)

mean
(Range )

ACCEPTED MANUSCRIPT
Moderate and

106.8

severe HIE

(60.5-108.1)

[88]

Serum
Control

20

ELISA

(1d)

(ng/ml)

4.15
(0.95)

Mild HIE

4.28
(0.63)

MBP
Hu SJ,et al

Serum
Control

21

Mild HIE

[2009]

[2007]

Control

(0.55)

7.07
(2.38)
7.97
(2.67)

Serum

10

ELISA (0-4d) 10-14d


( ug/L)

5.49
(8.27)

10

ModerateHIE 10

Severe HIE

(2.10)

(0.50)

AC

Mild HIE

4.85

1.50

TE

CE
P

[24]

6.88

(1.42)

Sun YL, et al.

(1.05)

(ug/L) 1.67

10

severe HIE

(0.58)

(1d)

ModerateHIE

ELIS

MA

[23]

4.67

NU

Severe HIE

5.40

(0.58)

SC
R

ModerateHIE 15

4.40

IP

[2009]

(7d)

Yuan EW, et al

5.88

5.78

(5.19)

(3.15)

5.98

5.95

(5.55 (2.73)
10

48.56

10.40

(16.89 (6.22)

S-100
Nagdyman
N, et al.

Control

20

Cord

IL

(24h)

blood

( ug/L)

0.8

[27]
[ 2001]

(0.7-1.0)
No or Mild HIE 22

1.5
(1.1-1.9)

Moderate and

2.5

severe HIE

Chen YM, et al.

(1.5-3.7)

Serum

ELISA

(1d)

ACCEPTED MANUSCRIPT
[29]

Control

50

(ug/L)

[2009]

1.163
(0.623)

Mild HIE

31

2.525

5.196

severe HIE

(2.479)

IP

Moderate and 29

GFAP
22

[33]
[2014]

Abnormal

Cord

ELISA

blood

(ng/ml)

[41]

Serum
Mild HIE

33

(0.08-0.6)

ELISA

(1d)

31

severe HIE
6

0.020
(0.090)
0.090
(0.006)

Poor prognosis

MA

(0.0003)

Moderate and

0.020

TE

Good prognosis 25

CE
P

UCH-L1
Chalak LF,et al.

Normal

22

Abnormal

(0.004)

Cord

ELISA

blood

(ng/ml)

1.88
(1.13 -3.24)

AC

[2014]

0.1

(ng/ml) 0.006

[2014]

[33]

(0.03-0.05)

NU

(HIE)

Jiang SH,et al.

0.03

SC
R

Chalak LF,et al. Normal

(1.261)

2.38

(HIE)

(1.90-3.00)

Douglas-Escobar
M, et al

Serum

Control

ELISA
ng/ml )

14

1.7
5.9-0.69

[40]
[2010]

HIE

14

2.8
(170.4-0.4)

Jiang SH,et al.


[41]

Serum
Mild HIE

33

[2014]

ELISA (1d)
(ng/ml) 1.2
(0.8)

Moderate and 31
severe HIE
Poor prognosis 6

2.3
(2.7)
0.8
(0.024)

Good prognosis 25

0.9

ACCEPTED MANUSCRIPT
(0.018)

BDNF

et al.

Normal

Cord

ELISA

blood

(pg/ml)

(674)
Severe

925

asphyxia

(513)

miRNA-21
Chen HJ, et al.

Serum

[52]

Control

PCR

29

(1d)

1.206

(0.103)

49

Activin A
Florio P, et al.

5.38

MA

HIE

NU

[2015]

CSF

10

TE

Moderate and

No or Mild HIE 20

(2004 )

severe HIE

AC

CE
P

Note: RID, Radioimmunoassay ;

chain reaction

SC
R

(2003)

[56]

1650

IP

[43]

1d

Chouthai NS,

(2.17)

ELISA

(1d)

(ug/L)

0.9
(0.04)
1.88
(0.14)

IL, immunoluminometric assay;

PCR, Polymerase

ACCEPTED MANUSCRIPT
Table 3
Summary of clinical studies investigating the cerebral blood vessel and blood brain barrier in

Clinical

marker

reference

grouping

Specimen Assay

Detection time

Median

Geometric

( Range)

mean

IP

Author and

(unit)

after birth ( h or d )

SC
R

Biological

neonates with HIE

Mean value

(Range )

(SD)

______________________________________________________________________________________________________________

MMP-9
Serum

Control

32

(ng/L)

[2013]
38

ModerateHIE 31

LIU H, et al.

[2009]

Control

CE
P

[59]

AC

HIE

(9.69)
46.41
(10.46)

ELISA

(1d)

(3d)

(ng/ml)

(7d)

99.36
(40.95)

82

492.19 713.34 438.60


(336.96) (304.16)(310.84)

Serum
Control

35.81

9.23

D
Serum

17

Jiang CY, et al.


[60]

(7.20)

(8.45)

25

TE

Severe HIE

28.87

MA

Mild HIE

3d

ELISA

NU

Huang HP, et al
[58]

20

ELISA (1d)

(3d)

(7d)

(pg/ml) 31.48

[2011]

(3.27)
HIE

40

31.44

37.50 33.42

(1.20) (4.46) (0.73)

VEGF
Chalak LF, et al.
[33]

Normal

22

Cord

ELISA

blood

(pg/ml)

243

[2014]

(137 -430)
Abnormal

1514

(HIE)

(456-5027)

Tan YF, et al.


[64]
[2004])

CSF
Control

13

ELISA
(pg/ml)

( 1d
10.94
(1.48)

ACCEPTED MANUSCRIPT
Mild HIE

16

12.30
(1.24)

ModerateHIE 13

13.60
(0.85)

14.79

Severe HIE

Serum

[ 64]

Control

13

[2004]

Mild HIE

16

ELISA
(pg/ml)

( 1d)

SC
R

Tan YF, et al.

IP

(1.63)

326.34

(64.40)

ModerateHIE

13

327.23

Severe HIE

NU

(80.54)

372.51

Shi X, et al.

MA

(90.94)

Serum

[65]

Control

20

[2006]

18

TE

Mild HIE

ModerateHIE

CE
P

Severe HIE

13

ELISA

(pg/ml)

(1d)

(2d)

(7d)

134.36 135.70 130.11


(52.27) (44.18) (38.24)
157.35 136.62 132.07
(65.01) (57.43) (36.24)
238.46 158.46 133.35
(85.33) (72.33) (45.86)
287.08 198.16 141.22
(92.61) (78.31) ( 68.70)

AC

_______________________________________________________________________________

ACCEPTED MANUSCRIPT
Table 4
Summary of clinical studies investigating the oxidative stress markers in neonates with HIE
Biological

Author and

Clinical

marker

reference

grouping

Specimen Assay
(unit)

Detection time

Median

after birth( h or d)

( Range)

mean
(Range )

Mean value

Geometric

(SD)

IP

______________________________________________________________________________________________________________

Cui YC, et al.

Serum

[69]

HIE

XO

26

(ug/ml)

[2012]

SC
R

SOD
(1d)

(3d)

(7d)

61.5

72.0 108.5

(12.4) (13.3) (14.1)

[71]

Serum
Control

40

XO

(2d)

NU

Wang JX, et al

(ug/ml)

(11.54)

Mild HIE

12

Moderate and 14

85.74

(10.68)
70.56
(9.68)

severe HIE

MA

[2009]

88.64

TE

MDA
Mondal N, et al

[2009]

Control

40

CE
P

[68]

AC

blood

NIA

(2d)

(umol/L)

3.11
(0.82)

Asphyxia with 40

7.52

epilepsy

(1.06)

Wan ZT, et al.


[69]

Crod

Control

Crod
40

blood

TBA

(1d)

(umol/L) 1.61

[2001]

(0.17)
Mild HIE

10

1.68
(0.53)

Moderate and

4.05

severe HIE

(1.14)

Wang JX, et al
[71]

Serum
Control

40

[2009]

TBA

(1d)

(nmol/ml) 6.52
(0.64)

Mild HIE

18

7.44
(1.24)

Moderate and
Severe HIE

14

8.94
(1.46)

Note: XO, Xanthine oxidase assay ; NIA, No introduction assay;

TBA, Thiobarbituric acid

ACCEPTED MANUSCRIPT
assay
Table 5
Summary of clinical studies investigating the Inflammation markers in neonates with HIE
marker

reference

Clinical

Specimen Assay

grouping

detection time

(unit)

Median

after birth ( h or d)
Mean value

( Range)

Geometric
mean
(Range )

SC
R

(SD)

Author and

IP

Biological

_______________________________________________________________________________
Hs-CRP
Shang Y et al.
[74]

Serum
Control

74

HIE

74

(1d)

(mg/L)

0.51

(0.18)

NU

[2014]

RIA

11.93

(1.91)

MA

Poor prognosis

Good prognosis

[75]

Serum

TE

Tian J C, et al.
Control

[2012]

CE
P

HIE

IL-1

30

AC

Normal

(mg/L)

(2.14)
5.99
(0.99)

(1d)

(3d)

(7d)

0.93
(0.18)

45

8.92 20.17 0.93


(1.74) (4.65) (0.18)

Chalak LF, et al.


[33]

IRST

9.71

22

Cord

ELISA

blood

( pg/ml )

456

[2014]

(354-577)
Abnormal

1324
(533-3289)

Jiang SH, et al.


[41]

Serum ELISA (1d)


Mild HIE

33

(pg/ml) 486

[ 2014]

(34)
Severe HIE

31

492
(52)

Good prognosis

25

492
(32)

Poor prognosis

521
25

Oygr N, et al.

CSF

ELISA

(1d)

ACCEPTED MANUSCRIPT
[ 89 ]

Control

11

(pg/ml)

0.48

[1998]

(0.125-3.3)
HIE

14

3.7
(0.125-7.4)

Cord

[33]

Normal

22

ELISA

blood

( pg/ml )

IP

Chalak LF,et al.

IL-6

[2014]
5

SC
R

Abnormal

Jiang SH, et al

(24 -165)
5404
(89-326766)

Serum ELISA (1d)


Mild HIE

33

(pg/ml) 17.4

NU

[41]

62

[2014]

(2.5)

Moderate and 31

371.6

(32.7)

MA

severe HIE
Poor prognosis 6

42
(3)

TE

Good prognosis 25

97.
(5)

Shang Y, et al.
[74]

Control

CE
P

[2014]

Serum

74

HIE

ELISA

(1d)

(pg/ml)

9.18
(1.27)

74

39.94
(4.46)

Poor prognosis

37.75

AC

4.22

Good prognosis

19.59
2.94

IL-8
Chalak, LF, et al.
[33])

Normal

Cord
22

ELISA

blood ( pg/ml )

154

[2014]

(98-240)
Abnormal

2535
295-21801

Jiang SH, et al
[74]

Serum
Mild HIE

33

[2014]

ELISA
(pg/ml)

(1d)
167.7
(27.4)

Moderate and
severe HIE

31

455.8
(45.9)

ACCEPTED MANUSCRIPT
Poor prognosis

144
(8)

Good prognosis

25

73

Serum ELISA

[81]

Control

24

(3d)

(pg/ml )

[2009]

(7d)

117.15 80.94

IP

Lu F, et al.

(10)

(139.15) (67.69)
8

304.51 173.73

SC
R

Mild HIE

(219.18)(107.12)

ModerateHIE

15

547.36 406.14

(285.88) (188.75)

Serum ELISA

Control

13

[2012]

Lu F, et al.
[81]

Control

CE
P

[2009]

TE

IL-10

Mild HIE

AC

ModerateHIE

Severe HIE

Control

Serum

24

(1-24h)(24-48h)
16.099

(8.528)(12.653)
46.92

33.800

(39.270)(23.381)

ELISA

(3d)

(pg/ml )

(7d)

14.57 13.71
(0.952) (0.866)

19.56 16.95
(1.95) (2.21)

15

26.58 18.43
(9.35) (4.83)

29.96 23.09
(2.86) (3.54)

Wang YC,et al.


[83]

(342.30) (274.69)

(pg/ml) 13.190

HIE with epilepsy 15

MA

Youn YA, et al
[82]

793.24 480.93

NU

Severe HIE

Serum
20

ELISA
(pg/ml)

153.0

[2009]

(80 -256)
HIE

20

316.0
(142-599)

IL-18
Guo YH, et al.
[87]

Control

Serum
20

[2014]

ELISA

(1d)

(3d)

(7d)

(ng/L)

71.08

72.53 71.93

(11.52) (11.05) (11.30)


Mild HIE

16

72.50

76.34 72.39

(4.05) (3.60) (6.02)


ModerateHIE

16

130.31 149.35 87.81

ACCEPTED MANUSCRIPT
(9.36) 914.359.72
159.59 189.06 106.78
(15.53)

Yuan EW, et al
[88]

Serum

Control

20

1d

ELISA

(pg/ml) 18.56

[2009]

(3.51)
9

ModerateHIE

19.63

15

(2.49)

22.80

20.30

(2.17)

(1.90

27.05

20.38

(5.56)

Chalak, LF, et al.

Cord

Normal

22

[2014]

Shang Y, et al.

[2014]

Control

Serum

74

HIE

(4-11)
24.3
13-44

ELISA

( 1 d)

(pg/ml) 17.20
(1.26)

74

97.00
(5.97)
90.23

AC

Poor prognosis

7.37

Good prognosis

44.32
4.84

Jiang SH, et al
[41]

6.6

CE
P

[74]

ELISA

blood ( pg/ml )

Abnormal

TE

(2014)

(3.08)

MA

TNF-a

[33]

19.67

(4.05)

NU

Severe HIE

(7d)

SC
R

Mild HIE

17.20
12.08

16

IP

Severe HIE

Serum ELISA
Mild HIE

33

(pg/ml)

[2014]

(1d)
15.3
(1.9)

Moderate and

31

11.1

severe HIE
Poor prognosis

(2.0)
6

13.3
(0.8)

Good prognosis 25

7.1
1.2

Tian JC,et al.


[75]

Serum
Control

30

ELISA
(mg/L)

(1d)
0.57

(2d) (7d)

ACCEPTED MANUSCRIPT
[2012]

(0.11)
HIE

45

1.15 1.87

0.84

(0.23) (0.35) (0.16)

[89]

CSF
Control

11

ELISA

37

Oygr N, et al.

(pg/ml)

37

(15.6-248)

14

Wang CQ

Serum

[92 ]

Control

23

ELISA
(ng/L)

(3d)

(7d)

42.5 44.8

(8.3) (6.8)

Mild HIE

NU

[2010]

SC
R

HIE

IP

[1998]

16

24

86.5 51.7

10

(11.3)

(5.8)

110.5 62.8
(9.2)

(12.5)

ICAM-1
Guo WY ,et al

[2011]

Control

Serum

50

CE
P

[94]

TE

Severe HIE

(6.7) (4.5)

MA

Moderate and

72.5 48.8

Mild HIE

AC

Moderate and

Severe HIE

(ng/ml)

53.35

15

98.94

17

167.87

(21.05)

(35.56)
13

217.58
(42.19)

Serum
Control

(3d)

(12.42)

Yi P, et al.
[95]

ELISA

40

ELISA
(ng/ml)

[2014]

(24h)
196.73
(5.85)

Mild HIE

15

219.37
(8.64)

Moderate and

21

510.03
(9.76)

Severe HIE

17

719.25
(10.13)

P-Selectin
Sun YZ, et al

Serum ELISA

(12h)

(48h)

(72h)

63.5
(27-468)

ACCEPTED MANUSCRIPT
[100]

Control

20

(ng/ml)

[2007]

146.47 134.36 135.70


(58.73) (52.27) (24.78)

Mild HIE

18

252,71 157.35 136.64


(73.95) (65.01) (57.43)

33

307.23 287.08 148.46

Moderate and

(85.64) (92.61) (72.33)


12

473.50 134.36 168.16

IP

Severe HIE

Cheng DJ et al.
[101]

Serum ELISA

Control

15

(ng/ml)

(3d)

71.81

(11.46)

Mild HIE

NU

[2006]

SC
R

(107.21)52.2778.31

15

295.05

(61.54)

Severe HIE

12

13

346.53

MA

Moderate and

(49.00)
472.55
(41.51)

_______________________________________________________________________________

AC

CE
P

TE

Note: RIA, Radioimmunity assay IRST, Immune rate scattering turbidity assay

ACCEPTED MANUSCRIPT

Table 6
Summary of clinical studies investigating the metabolism markers in neonates with HIE
Clinical

marker

reference

grouping

Specimen Assay
(unit)

Detection time
after birth ( h or d )
Mean value

Geometric

( Range)

SC
R

(SD)

Median

Author and

IP

Biological

mean
(Range )

_______________________________________________________________________________
LDH
Zhang JX, et al.
[103]

Serum

Control

30

HIE

66

( u/L

98.33 98.33

181.2 68.3

MA

[104]

Serum

Good outcome

TE

Beken S, et al.

Serum

CE
P

I stage HIE

29

( 662-1866)

NIA
( IU/L )

36

649

1015
(355-5252)
1729

AC

29

(58-7865)

Hayakawa M, et al.
[104]

(507-1204)

(348-1096)

II stage HIE

III stage HIE

673

987

Poor outcome

(23.8) (18.6)

NIA

( IU/L )

[2014]

CK-BB

(7d)

(18.69) (18.69)

Hayakawa M, et al.

[2014]

(3d)

NU

[2015]

[105]

CL

Serum

Good outcome

NIA
( IU/L )

[2014]

642
(433-1328)

Poor outcome

1022
(538-2603)

Beken S, et al.
[105]

Serum NIA
Stage I HIE

29

[2014]

(mg/dl )

617
(160-3480)

Stage II HIE

36

2159
(49-13092)

Stage III HIE

29

2664
(238-39252)

ACCEPTED MANUSCRIPT

Nagdyman N, et al
[112]

Cord

Control

20

blood

IF

(2h)

(ug/L )

10.0

[ 2001]

(6.0-13.0)
16.0

No or Mild HIE 22

(13.0-23.5)

IP

Moderate and

Glutamate
Zhang CY, et al.
[115]

Serum

Control

30

Pigtags

46.5

(21.4-83.0)

SC
R

severe HIE

(1d)

(3d)

(7d)

(umol/ml) 69.26
(9.54)

HIE

NU

(2010)
30

78.32 95.33 72.36

MA

(15.49) (19.72) (12.23)

Hagberg H, et al.
[116]

Moderate HIE

TE

Severe HIE

NIA

(1d)

( mumol/L) 2.7

[1993]

CSF

Mild HIE

3.2
12.3

_______________________________________________________________________________

CE
P

assay

CL, Chemiluminescence assay; IF, Immunofluorescence assay;

AC

Note:

NIA, No introduction

ACCEPTED MANUSCRIPT
Highlights
Biomarker application in neonatal hypoxic ischemic encephalopathy is introduced

IP

S-100 and activin A are good markers for early diagnosis of neonatal HIE

SC
R

GFAP and UCH-L1 are promising biomarkers to predict the long-term neurologic handicap

AC

CE
P

TE

MA

NU

Optimized combination of biomarkers is proposed to enhance neonatal HIE diagnosis