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Neonatal hypoxic ischemic encephalopathy-related biomarkers in serum and cerebrospinal fluid
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
To appear in:
Clinica Chimica Acta
Received date: Revised date: 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 fluid, Clinica Chimica Acta (2015), doi: 10.1016/j.cca.2015.08.021
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ACCEPTED MANUSCRIPT Neonatal hypoxic ischemic encephalopathy-related biomarkers in serum and cerebrospinal fluid 1
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, Lihong Yang , Pengshun Ren , Yihui Yang ,
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Jinsheng Gao , Lianxiang Li
1，2
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, Qiuli Wang , Sujing Wu 2,3
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1，2
Hongyan Lv
1
Department of Neonatology, Handan Maternal and Child Care Centers, Handan 2
056002,Hebei Province, P.R. China; Department of Neonatal pathology, Handan Maternal 3
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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, 4
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: [email protected] Tel: + 86 3106038483; + 86 3102116095
ACCEPTED MANUSCRIPT 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
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mechanism of neonatal hypoxic ischemic encephalopathy in relation to numerous
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brain-related biomarkers including NSE, S-100β, GFAP, UCH-L1, Tau protein, miRNA, LDH,
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and CK-BB. In early diagnosis of neonatal HIE, S-100β 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
ACCEPTED MANUSCRIPT 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-α, Tumor necrosis factor-α; LDH, Lactic dehydrogenase; CK-BB, Creatine
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kinase BB.
ACCEPTED MANUSCRIPT 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
a
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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1.14–11.7%, and the incidence of HIE in full-term live birth infants is 1–2/1000 of affected newborns. Approximately 15–20% of affected newborns will succumb within the neonatal
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period, and an additional 25–30% 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 child’s 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
ACCEPTED MANUSCRIPT 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
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Brain oxygen consumption accounts for 20–25% 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 or”reperfusion 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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,
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recovery effects and when to end treatment.
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4. Neonatal HIE–related 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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(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-100β 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-100β are brain specific and the most important and most active members of the central nervous
ACCEPTED MANUSCRIPT system.Proteins S-100β are synthesized and secreted by astrocytes and Schwann cell. Under normal circumstances, there are small amounts of protein S-100β 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 S100β into the blood and cerebrospinal fluid. Protein S-100β 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-100β greater than 2.02
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µg/L, with a sensitivity of 86.7% and specificity of 88% predicted moderate and severe
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HIE[25]. Because protein S-100β are secreted by astrocytes, the detection of serum concentrations protein S-100β can also be used to determine blood brain barrier damage
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[26] or central nervous system inflammation. The protein S-100β 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 S100β gradually increased at 0.5–1 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-100β release, and it is a direct marker of neuronal brain damage when elevated concentrations of S-100β are detected in plasma or cerebrospinal fluid. Higher concentration of S-100β are observed in umbilical cord blood from perinatal asphyxia infants and neonatal HIE patients, serum concentration S-100β of 8.5 µg/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-100β 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-100β can be detected in
ACCEPTED MANUSCRIPT saliva to predict neural function abnormalities caused by neonatal asphyxia. The study showed that asphyxia neonatal neurological prognosis was poor in children when S-100β
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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-100β achieved a sensitivity of 100% (confidence interval [CI]5-95%: 89.3–100.0%), and specificity of 100% (CI5-95%: 98.6–100%) 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-100β is a promising biomarker
4.1.4 Glial fibrillar acidic protein (GFAP）
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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-100β, 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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: 0.687–0.742] [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 Brain–derived 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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 β (TGF-β) 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
ACCEPTED MANUSCRIPT 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 µg/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: 69.0–100%] and 100% [95%CI: 94.3–100%][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 µg/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
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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-100β, 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
ACCEPTED MANUSCRIPT 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],
ACCEPTED MANUSCRIPT 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
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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 -2
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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
ACCEPTED MANUSCRIPT 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
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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 Inflammation–related 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].
ACCEPTED MANUSCRIPT 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)-α 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-α, 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
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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-1β promotes brain damage in the central nervous system. The mechanism of brain damage induced by IL-1β involves the release of free radicals, stimulating inflammatory reactions and enhancing the toxicity of excitatory amino acids. Relevant data show that cord
ACCEPTED MANUSCRIPT blood and peripheral venous blood of IL-1β, IL-8, and TNF-α concentrations in neonatal HIE patients were higher than in the control group, and that serum IL-1β concentrations and
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neonatal HIE scores were positively correlated. Elevated levels of serum IL-1β 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-1β , IL-6, and TNF-α concentrations were significantly higher than in the control group,
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demonstrating that cerebrospinal fluid IL-1β was closely related to the diagnosis of neonatal
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HIE[78].Therefore, the detection of cerebrospinal fluid IL-1β concentrations can help determine the prognosis of neonatal HIE patients.
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IL-6 mainly produced by glial cell inhibits the synthesis of TNF-α and IL-1β, 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
ACCEPTED MANUSCRIPT enhanced IL-1β 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.
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IL-10 plays a protective role in brain tissue by inhibting the secretion of IL-1β, 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-1β 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
ACCEPTED MANUSCRIPT 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-α
TNF-a promotes the synthesis and release of IL-1β and IL-8, the induction of apoptosis of nerve cells, disruption of the blood brain barrier, and aggravates brain damage. Oygür 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.
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Cerebrospinal fluid levels of IL-6 and TNF-α were significantly higher in the abnormal group
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compared with the normal group, and IL-6 was a better than TNF-α 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-α concentration was closely
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related to the severity of HIE and the prognosis of the patients[90-92]. Serum TNF-α represents inflammatory reactions of many tissues and organs after neonatal HIE, whereas TNF-α concentrations in the cerebrospinal fluid are limited to brain tissue; therefore, the detection of TNF-α 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
ACCEPTED MANUSCRIPT 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-1β, TNF-α, interferon-γ 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
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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.
ACCEPTED MANUSCRIPT
4.4.5 Selectins early
stage
of
ischemic
cerebral
vascular
disease,
selectins
T
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
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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].
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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-100β combined with GAFP, S-100β 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
ACCEPTED MANUSCRIPT 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-100β 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
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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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Neonate 2001;79:224–27
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[81] Lu F, Yang MH, Qian ZX. Clinical Changes significance of change of serum interleukin-8 and interleukin -10 level in neonatal hypoxic ischemic encephalopathy. Heilongjiang Medical Journal 2009;33:641-43 [82] Youn YA, Kim SJ, Sung IK, et al. Serial examination of serum IL-8, IL-10 and IL-1Ra levels is significant in neonatal seizures induced by hypoxic-ischaemic encephalopathy. Scand J immunol 2012;76:286-93 [83] Wang YC, Shi CC, Ni H. Clinical significance of serum interleukin 10 levels in neonatal hypoxic ischemic encephalopathy. J Applied Clincal Pediatrics 2003;18:442-43 [84] Róka A, Bekǒ G, Halósz J,et al. Changes in serum cytokine and cortisol levels in
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T
[85] Felderhoff-Mueser U, Schmidt Ol, Oberholzer A, et al. IL-18: a key player in
SC R
neuroinflammation and neurodegenneration.Trends Neurosci 2005; 28:487-493 [86] Guo J, Wang GT, Lin L, et al. The changes of serum IL-18,IL-6 and IFN-r in newborns with hypoxic-ischemic encephalopathy. J Xiangnan University(Medical Sciences) 2014;16:11-13
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[87] Guo YH, Li GX, Li WH, Liu ZS, Bai XM. Dynamic study of serum interleukin 18 levels in
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neonatal hypoxic ischemic encephalopathy. J Hebei Medical University 2014;35:1337-39 [88] Yuan EW, YU WQ, Yang M, et al. Detection and clincal significance of IL-18 and
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TE
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D
neuron-specific enolase is serum of newborns with hypoxic-ischemic encephalopathy.
CE P
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infants with hypoxic-ischaemic encephalopathy. Arch Dis Child Fetal Neonatal 1998; 79:F190-93
[90] Silveura RC, Procianoy RS.Interleukin-6 and tumor necrosis factor-alpha levels in plasma and cerebrospinal fluid of term newborn infants with hypoxic-ischemic encephalopathy. J Pediatr 2003；143:625-29 [91]Ceccon ME. Interleukins in hypoxic-ischemic encephalopathy. J Pediatr Rio J 2003;79:280-81 [92]Wang CQ. Investgation on the changes of TNF-a in neonatal hypoxic ischemic encephalopathy. Maternal and Child Health Care China 2010; 25:543-45
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cells in Fas/FasL interation. Immunotherapy 2007;30:727-39
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SC R
[94] Guo WY, Li RS, Wang W,Dong DB, Wang AY.Expression and clinical significance of hypoxic-ischemic encephalopathy. Progress in Modern
Biomedicine 2011;11:3135-37
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[95] Yi B, Liu KL, Shen H. Changes of serum sICAM-1 and NSE levels in hypoxic ischemic
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encephalopathy. Shandong Medical J 2014;54:54-55
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[97] Ley K, Bullard DC, Arbones ML, et al. Sequential contribution of L- and P-selectin to
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leukocyte rolling in vivo. J Exp Med 1995;181:669-75. [98] Grosshaupt B, Muntean W, Sedlmayr P. Hyporeactivity of neonatal platelets is not caused
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ACCEPTED MANUSCRIPT [102] Song JZ, Zhou ZY, Li ZG, et al. The significance of lymphocyte L-selectin and ICAM-1 levels
in
newborns
with
hypoxic-ischemic
encephalopathy.Chinese
Neonatology
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2008;23:75-77
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[103] Zhang JX, Dong L. detection of serum LDH in neonatal encephalopathy. J Inner Monglia Medical University 2015;37:72-73
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deficiency for creatine kinases BCK and UbCKmit. Brain Res 2005;157:219-34
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[107] Walsh P, Jedeikin R, Ellis G, et al. Assessment of neurologic outcome in asphyxiated term infants by use of serial CK-BB isoenzyme measurement. J Pediatri1982;101:988-92
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[108] Fernandez F, Verdu A, Quero J,et al. Serum CPK-BB isoenzyme in the assessment of brain damage in asphyctic term infants. Acta paediatri scand 1987; 76:914-18 [109] Sweet DG, Bell A H, McClure G,et al. Comparision between creatin kinase brain isoenzyme(CCBB) activity and Sarnat score for prediction of adverse outcome following perinatal asphyxia. J Perinatal Medicine1999;27:478-83 [110] Nagdyman N, Grimmer I,Scholz T, et al. Predictive value of brain-specific proteins in
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[112] Zhang AM, Liu CY, Lv ZF, Duan QJ. Cerrelation between serum CK-BB and cerebral CT
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value in asphyxiated term infant with hypoxic-ischemic encephalopathy.Chinese J Birth Health &Heredity 2006;14: 90-92
[113] Gücüyener K, Atalay Y, Aral YZ, et al. Excitatory amino acids and taurine levels in
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[114] Pu Y, Li QF, Zeng CM,et al. Increased detectability of alpha brain glutamataglutamine in
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[115] Zhang CY, Han HH, Ge QF. Dynamic changes of serum glutamate level in
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hypoxia-ischemia encephalopathy. China Practical Medicine 2010; 5:3-4 [116] Hagberg H, Thornberg E, Blennow M,et al. Excitatory amino acids in the cerebrospinal of
asphyxiated
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Paediatr1993; 82:925-29
infants:relationship
to
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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-100β﹡﹡﹡ ↑
D
matter
Y
Brain injury
death astrocytes
or poor
but can released
prognosis
References
(%）
Severe HIE:
Severe HIE:
79%
70%
NU
MBP﹡
↑
MA
NSE﹡﹡
Specificity
IP
Biomarker
T
summary of neonatal hypoxic ischemic encephalopathy related biomarkers
[13-17】
Poor prognosis Poor prognosis
IC
Y
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﹡﹡
Y
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
Y
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
Y
prediction
HIE：100%
HIE：100%
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
↑
D
ocytes, etc.
VEGF﹡
IC
NU
Released after
MA
↑
SC R
IP
injury
MMP-9﹡﹡
【56】
T
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-1β﹡﹡
↑
Released after
Acute phase
astrocytes
HIE, severe
vascular endothelial
brain prognosis
Y
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
D
↑
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﹡﹡
T
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
Y
79%
56%
ACCEPTED MANUSCRIPT
CK-BB﹡﹡﹡ ↑ Released after neuron, glial cells , placenta
Severe brain
Y
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
T
lung,iKidney,
vessel;
NU
Note: HIE, neonatal hypoxic ischemic encephalopathy； CSF, Cerebrospinal fluid; BBB, Blood brain barrier；PVH, 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,
D
MicroRNA; MMP-9, Matrix metalloproteinase-9; VEGF, Vascular endothelial growth factor;
CE P
protein; IL-1β, Interleukin-1β; 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
n
Specimen Assay (unit)
Detection time After birth ( h or d) Mean value
NSE Liu CY, et al
Serum
[14]
Normal
7
Mild HIE
12
RID (ng/ml)
( Range)
(1d)
3.92
(1.47)
NU
[ 2005]
Geometric
SC R
(SD) ———————————————————————————————————————————————————————
Median
T
Author and
IP
Biological
8.42
ModerateHIE 14
9
D
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
7
ELISA
（1d） .
(ug/L)
1.33
[1997]
(0.72) Mild HIE
7
1.45 (1.42)
ModerateHIE
7
4.21 (2.70)
Severe HIE
7
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
7
106.8
severe HIE
(60.5-108.1)
[88]
Serum Control
20
ELISA
(1d)
(ng/ml)
4.15 (0.95)
Mild HIE
9
4.28 (0.63)
8
MBP Hu SJ,et al
Serum Control
21
Mild HIE
5
[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)
6
Sun YL, et al.
(1.05)
(ug/L) 1.67
10
severe HIE
(0.58)
(1d)
D
ModerateHIE
ELIS
MA
[23]
4.67
NU
Severe HIE
5.40
(0.58)
SC R
ModerateHIE 15
4.40
IP
[2009]
(7d)
T
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-100β 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
7
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)
5
[41]
Serum Mild HIE
33
(0.08-0.6)
ELISA
(1d)
31
severe HIE 6
0.020 (0.090) 0.090 (0.006)
D
Poor prognosis
MA
(0.0003)
Moderate and
0.020
TE
Good prognosis 25
CE P
UCH-L1 Chalak LF,et al.
Normal
22
Abnormal
5
(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
T
(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
D
No or Mild HIE 20
(2004 )
severe HIE
AC
CE P
Note: RID, Radioimmunoassay ;
chain reaction
SC R
(2003)
[56]
1650
IP
[43]
（1d）
T
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
n
Specimen Assay
Detection time
Median
Geometric
( Range)
mean
IP
Author and
(unit)
after birth ( h or d )
SC R
Biological
T
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
5
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)
9
14.79
T
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)
9
372.51
Shi X, et al.
MA
(90.94)
Serum
[65]
Control
20
[2006]
D
18
TE
Mild HIE
ModerateHIE
CE P
Severe HIE
13
9
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
n
Specimen Assay (unit)
Detection time
Median
after birth( h or d)
( Range)
mean (Range )
T
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)
D
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
9
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
n
Specimen Assay
grouping
detection time
(unit)
Median
after birth ( h or d) Mean value
( Range)
Geometric mean (Range )
SC R
(SD)
T
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
D
Good prognosis
[75]
Serum
TE
Tian J C, et al. Control
[2012]
CE P
HIE
IL-1β
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
5
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
6
521 （25）
Oygür 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.
T
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)
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D
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
5
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
6
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.
T
(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)
9
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)
8
19.56 16.95 (1.95) (2.21)
15
26.58 18.43 (9.35) (4.83)
9
29.96 23.09 (2.86) (3.54)
Wang YC,et al. [83]
(342.30) (274.69)
(pg/ml) 13.190
D
HIE with epilepsy 15
MA
Youn YA, et al [82]
793.24 480.93
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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) 914.35）（9.72） 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
8
(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
5
CE P
[74]
ELISA
blood ( pg/ml )
D
Abnormal
TE
(2014)
(3.08)
MA
TNF-a
[33]
19.67
(4.05)
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Severe HIE
(7d)
SC R
Mild HIE
17.20） （12.08）
T
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
T
Oygür 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
D
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
T
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)（52.27）（78.31）
15
295.05
(61.54)
Severe HIE
12
13
346.53
MA
Moderate and
(49.00) 472.55 (41.51)
D
_______________________________________________________________________________
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
n
Specimen Assay (unit)
Detection time after birth ( h or d ) Mean value
Geometric
( Range)
SC R
(SD)
Median
T
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
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[104]
Serum
Good outcome
TE
Beken S, et al.
Serum
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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
D
Poor outcome
(23.8) (18.6)
NIA
( IU/L )
[2014]
CK-BB
(7d)
(18.69) (18.69)
Hayakawa M, et al.
[2014]
(3d)
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[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
T
No or Mild HIE 22
(13.0-23.5)
7
IP
Moderate and
Glutamate Zhang CY, et al. [115]
Serum
Control
30
Pigtag’s
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
D
[1993]
CSF
Mild HIE
3.2 12.3
_______________________________________________________________________________
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assay
CL, Chemiluminescence assay; IF, Immunofluorescence assay;
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Note:
NIA, No introduction
ACCEPTED MANUSCRIPT Highlights 。Biomarker application in neonatal hypoxic ischemic encephalopathy is introduced
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。S-100β and activin A are good markers for early diagnosis of neonatal HIE
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。GFAP and UCH-L1 are promising biomarkers to predict the long-term neurologic handicap
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CE P
TE
D
MA
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。Optimized combination of biomarkers is proposed to enhance neonatal HIE diagnosis
S0009-8981(15)00401-5 doi: 10.1016/j.cca.2015.08.021 CCA 14083
To appear in:
Clinica Chimica Acta
Received date: Revised date: 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 fluid, Clinica Chimica Acta (2015), doi: 10.1016/j.cca.2015.08.021
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ACCEPTED MANUSCRIPT Neonatal hypoxic ischemic encephalopathy-related biomarkers in serum and cerebrospinal fluid 1
1
3
, Lihong Yang , Pengshun Ren , Yihui Yang ,
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Jinsheng Gao , Lianxiang Li
1，2
T
1
, Qiuli Wang , Sujing Wu 2,3
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1，2
Hongyan Lv
1
Department of Neonatology, Handan Maternal and Child Care Centers, Handan 2
056002,Hebei Province, P.R. China; Department of Neonatal pathology, Handan Maternal 3
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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, 4
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: [email protected] Tel: + 86 3106038483; + 86 3102116095
ACCEPTED MANUSCRIPT 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
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mechanism of neonatal hypoxic ischemic encephalopathy in relation to numerous
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brain-related biomarkers including NSE, S-100β, GFAP, UCH-L1, Tau protein, miRNA, LDH,
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and CK-BB. In early diagnosis of neonatal HIE, S-100β 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
ACCEPTED MANUSCRIPT 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-α, Tumor necrosis factor-α; LDH, Lactic dehydrogenase; CK-BB, Creatine
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kinase BB.
ACCEPTED MANUSCRIPT 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
a
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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1.14–11.7%, and the incidence of HIE in full-term live birth infants is 1–2/1000 of affected newborns. Approximately 15–20% of affected newborns will succumb within the neonatal
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period, and an additional 25–30% 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 child’s 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
ACCEPTED MANUSCRIPT 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
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Brain oxygen consumption accounts for 20–25% 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 or”reperfusion 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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,
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recovery effects and when to end treatment.
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4. Neonatal HIE–related 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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(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-100β 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-100β are brain specific and the most important and most active members of the central nervous
ACCEPTED MANUSCRIPT system.Proteins S-100β are synthesized and secreted by astrocytes and Schwann cell. Under normal circumstances, there are small amounts of protein S-100β 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 S100β into the blood and cerebrospinal fluid. Protein S-100β 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-100β greater than 2.02
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µg/L, with a sensitivity of 86.7% and specificity of 88% predicted moderate and severe
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HIE[25]. Because protein S-100β are secreted by astrocytes, the detection of serum concentrations protein S-100β can also be used to determine blood brain barrier damage
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[26] or central nervous system inflammation. The protein S-100β 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 S100β gradually increased at 0.5–1 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-100β release, and it is a direct marker of neuronal brain damage when elevated concentrations of S-100β are detected in plasma or cerebrospinal fluid. Higher concentration of S-100β are observed in umbilical cord blood from perinatal asphyxia infants and neonatal HIE patients, serum concentration S-100β of 8.5 µg/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-100β 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-100β can be detected in
ACCEPTED MANUSCRIPT saliva to predict neural function abnormalities caused by neonatal asphyxia. The study showed that asphyxia neonatal neurological prognosis was poor in children when S-100β
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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-100β achieved a sensitivity of 100% (confidence interval [CI]5-95%: 89.3–100.0%), and specificity of 100% (CI5-95%: 98.6–100%) 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-100β is a promising biomarker
4.1.4 Glial fibrillar acidic protein (GFAP）
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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-100β, 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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: 0.687–0.742] [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 Brain–derived 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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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 β (TGF-β) 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
ACCEPTED MANUSCRIPT 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 µg/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: 69.0–100%] and 100% [95%CI: 94.3–100%][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 µg/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
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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-100β, 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
ACCEPTED MANUSCRIPT 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],
ACCEPTED MANUSCRIPT 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
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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 -2
-
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
ACCEPTED MANUSCRIPT 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
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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 Inflammation–related 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].
ACCEPTED MANUSCRIPT 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)-α 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-α, 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
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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-1β promotes brain damage in the central nervous system. The mechanism of brain damage induced by IL-1β involves the release of free radicals, stimulating inflammatory reactions and enhancing the toxicity of excitatory amino acids. Relevant data show that cord
ACCEPTED MANUSCRIPT blood and peripheral venous blood of IL-1β, IL-8, and TNF-α concentrations in neonatal HIE patients were higher than in the control group, and that serum IL-1β concentrations and
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neonatal HIE scores were positively correlated. Elevated levels of serum IL-1β 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-1β , IL-6, and TNF-α concentrations were significantly higher than in the control group,
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demonstrating that cerebrospinal fluid IL-1β was closely related to the diagnosis of neonatal
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HIE[78].Therefore, the detection of cerebrospinal fluid IL-1β concentrations can help determine the prognosis of neonatal HIE patients.
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IL-6 mainly produced by glial cell inhibits the synthesis of TNF-α and IL-1β, 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
ACCEPTED MANUSCRIPT enhanced IL-1β 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.
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IL-10 plays a protective role in brain tissue by inhibting the secretion of IL-1β, 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-1β 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
ACCEPTED MANUSCRIPT 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-α
TNF-a promotes the synthesis and release of IL-1β and IL-8, the induction of apoptosis of nerve cells, disruption of the blood brain barrier, and aggravates brain damage. Oygür 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.
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Cerebrospinal fluid levels of IL-6 and TNF-α were significantly higher in the abnormal group
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compared with the normal group, and IL-6 was a better than TNF-α 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-α concentration was closely
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related to the severity of HIE and the prognosis of the patients[90-92]. Serum TNF-α represents inflammatory reactions of many tissues and organs after neonatal HIE, whereas TNF-α concentrations in the cerebrospinal fluid are limited to brain tissue; therefore, the detection of TNF-α 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
ACCEPTED MANUSCRIPT 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-1β, TNF-α, interferon-γ 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
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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.
ACCEPTED MANUSCRIPT
4.4.5 Selectins early
stage
of
ischemic
cerebral
vascular
disease,
selectins
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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
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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].
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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
ACCEPTED MANUSCRIPT 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
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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-100β combined with GAFP, S-100β 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
ACCEPTED MANUSCRIPT 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-100β 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
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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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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
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S-100β﹡﹡﹡ ↑
D
matter
Y
Brain injury
death astrocytes
or poor
but can released
prognosis
References
(%）
Severe HIE:
Severe HIE:
79%
70%
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MBP﹡
↑
MA
NSE﹡﹡
Specificity
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Biomarker
T
summary of neonatal hypoxic ischemic encephalopathy related biomarkers
[13-17】
Poor prognosis Poor prognosis
IC
Y
84 %
70%
UK
UK
Severe HIE: 83%-86.7%
Severe HIE:
[23,24】
[25,27-29】
88%-95%
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from other tissue damage
↑
Released after
Severe brain
death astrocytes
injury or poor
AC
GFAP﹡﹡
Y
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
Y
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
Y
prediction
HIE：100%
HIE：100%
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]
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↑
D
ocytes, etc.
VEGF﹡
IC
NU
Released after
MA
↑
SC R
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injury
MMP-9﹡﹡
【56】
T
neuron injury
prediction
astrocytes,
process of
microglia cells
HIE,
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markers
pathological
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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-1β﹡﹡
↑
Released after
Acute phase
astrocytes
HIE, severe
vascular endothelial
brain prognosis
Y
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
D
↑
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﹡﹡
T
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
Y
79%
56%
ACCEPTED MANUSCRIPT
CK-BB﹡﹡﹡ ↑ Released after neuron, glial cells , placenta
Severe brain
Y
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
T
lung,iKidney,
vessel;
NU
Note: HIE, neonatal hypoxic ischemic encephalopathy； CSF, Cerebrospinal fluid; BBB, Blood brain barrier；PVH, 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,
D
MicroRNA; MMP-9, Matrix metalloproteinase-9; VEGF, Vascular endothelial growth factor;
CE P
protein; IL-1β, Interleukin-1β; 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
n
Specimen Assay (unit)
Detection time After birth ( h or d) Mean value
NSE Liu CY, et al
Serum
[14]
Normal
7
Mild HIE
12
RID (ng/ml)
( Range)
(1d)
3.92
(1.47)
NU
[ 2005]
Geometric
SC R
(SD) ———————————————————————————————————————————————————————
Median
T
Author and
IP
Biological
8.42
ModerateHIE 14
9
D
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
7
ELISA
（1d） .
(ug/L)
1.33
[1997]
(0.72) Mild HIE
7
1.45 (1.42)
ModerateHIE
7
4.21 (2.70)
Severe HIE
7
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
7
106.8
severe HIE
(60.5-108.1)
[88]
Serum Control
20
ELISA
(1d)
(ng/ml)
4.15 (0.95)
Mild HIE
9
4.28 (0.63)
8
MBP Hu SJ,et al
Serum Control
21
Mild HIE
5
[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)
6
Sun YL, et al.
(1.05)
(ug/L) 1.67
10
severe HIE
(0.58)
(1d)
D
ModerateHIE
ELIS
MA
[23]
4.67
NU
Severe HIE
5.40
(0.58)
SC R
ModerateHIE 15
4.40
IP
[2009]
(7d)
T
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-100β 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
7
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)
5
[41]
Serum Mild HIE
33
(0.08-0.6)
ELISA
(1d)
31
severe HIE 6
0.020 (0.090) 0.090 (0.006)
D
Poor prognosis
MA
(0.0003)
Moderate and
0.020
TE
Good prognosis 25
CE P
UCH-L1 Chalak LF,et al.
Normal
22
Abnormal
5
(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
T
(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
D
No or Mild HIE 20
(2004 )
severe HIE
AC
CE P
Note: RID, Radioimmunoassay ;
chain reaction
SC R
(2003)
[56]
1650
IP
[43]
（1d）
T
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
n
Specimen Assay
Detection time
Median
Geometric
( Range)
mean
IP
Author and
(unit)
after birth ( h or d )
SC R
Biological
T
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
5
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)
9
14.79
T
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)
9
372.51
Shi X, et al.
MA
(90.94)
Serum
[65]
Control
20
[2006]
D
18
TE
Mild HIE
ModerateHIE
CE P
Severe HIE
13
9
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
n
Specimen Assay (unit)
Detection time
Median
after birth( h or d)
( Range)
mean (Range )
T
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)
D
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
9
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
n
Specimen Assay
grouping
detection time
(unit)
Median
after birth ( h or d) Mean value
( Range)
Geometric mean (Range )
SC R
(SD)
T
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
D
Good prognosis
[75]
Serum
TE
Tian J C, et al. Control
[2012]
CE P
HIE
IL-1β
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
5
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
6
521 （25）
Oygür 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.
T
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
D
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
5
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
6
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.
T
(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)
9
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)
8
19.56 16.95 (1.95) (2.21)
15
26.58 18.43 (9.35) (4.83)
9
29.96 23.09 (2.86) (3.54)
Wang YC,et al. [83]
(342.30) (274.69)
(pg/ml) 13.190
D
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) 914.35）（9.72） 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
8
(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
5
CE P
[74]
ELISA
blood ( pg/ml )
D
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）
T
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
T
Oygür 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
D
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
T
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)（52.27）（78.31）
15
295.05
(61.54)
Severe HIE
12
13
346.53
MA
Moderate and
(49.00) 472.55 (41.51)
D
_______________________________________________________________________________
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
n
Specimen Assay (unit)
Detection time after birth ( h or d ) Mean value
Geometric
( Range)
SC R
(SD)
Median
T
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
D
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
T
No or Mild HIE 22
(13.0-23.5)
7
IP
Moderate and
Glutamate Zhang CY, et al. [115]
Serum
Control
30
Pigtag’s
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
D
[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
T
。S-100β 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
D
MA
NU
。Optimized combination of biomarkers is proposed to enhance neonatal HIE diagnosis