Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2018 15(4): 3718-3732. OPEN ACCESS

Serum Glial Fibrillary Acidic Protein and Cranial Computed Tomography in Neonates with Hypoxic Ischemic

Hala Goda Elnady1, Naglaa Abd El Moneam Abdallah2, Sonia Kamel Sayed2 Naglaa Mohamed Kholoussi3, Tarek Hassan Nabil Morsy,4Gehad Faisal Elzanaty,5and Rania Fawzy3

1Child health Dept* National Research Centre, Egypt. 2Pediatric Dept., Faculty of Medicine, Al Azhar University, Egypt. 3Immunogenetics Dept, National Research Centre, Egypt. 4Radiology Dept , Military Medical Academy, Egypt. 5Abo Homos Central Hospital, Ministry of Health. Egypt. . *Correspondence: [email protected] Accepted: 02 Nov 2018 Published online: 23 Dec. 2018 Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication, while numerous potential biomarkers of the brain damage can exist but Glial Fibrillary Acidic Protein (GFAP) hold a significant promise in this group, as it is a key protein in astrocytes that its expression is induced by brain damage, CT show characteristic patterns of brain injury which correlate with the level of brain maturity at the time of an insult, therefore we can exclude other causes of encephalopathy in addition limiting the diagnosis to HIE. Assessment of serum GFAP in neonate with HIE and its relation to grading and to evaluate the role of cranial CT in diagnosis of HIE and its relation to GFAP finding. A case control study was carried out on 30 full term neonates with HIE classified according to Sarnat and Sarnat classification and 20 full term healthy neonates, selected from Rashid General Hospital in Alexandria, Egypt. Samples for GFAP were obtained at two-points time (0-6 & 48-72) hours postnatal in cases, and for control within hours from birth, Cranial Computed Tomography (CT) were obtained at the 4th day age for cases group. The laboratory tests are done in the Immunogenetics Lab, National Research Centre, Dokki, Giza, Egypt. In our study there was statistically significant serial increase in GFAP level in cases of HIE than control from birth to 48-72 hours age and positive correlation between its level and severity of the disease, 26.7% of cases of HIE had a picture of brain edema in cranial CT and this finding also correlated with the level of GFAP. There is serial increase in serum GFAP level from birth to 48-72 hours postnatal in HIE and that increase is correlated with its severity, also brain edema is the most common finding in cranial CT in these cases and it is correlated with the severity and GFAP level. Keywords: hypoxic ischemic encephalopathy, glial fibrillary acidic protein, computed tomography

INTRODUCTION gestation which manifested by a subnormal level Neonatal encephalopathy stands a of consciousness or . In many cases, it is heterogeneous, clinically well-defined syndrome accompanied by difficulty with starting or categorized by disturbance in the neurological maintaining respiration and depression of tone function during the initial days of life in an in a and reflexes (Barnette et al.,2014). newborn who born at (or beyond) 35 weeks of Hypoxic ischemic encephalopathy (HIE) is a Sayed et al., Serum GFAP and CT in Neonates with Hypoxic Ischemic Encephalopathy. potentially upsetting condition accounts for 25% of neurological deficits), Cortical visual impairment or neonatal deaths besides, it contributes to blindness, Cerebral palsy (CP) in reported 10%- significant financial and personal burden, adding 13% of infants following moderate-to-severe HIE, to (10-15%) of infants affected by HIE mostly will spastic quadriplegia and dyskinetic CP are most die before discharge from the hospital, (10% to common types (Mwaniki et al., 2012). Other 15%) will develop cerebral palsy, and (40%) will complications as learning disabilities be affected by blindness, deafness, autism, characterized by lower working memory, and developmental delay, , or other long-term attention /executive function impairments, complications (Felipe et al., 2013). behavioral difficulties especially hyperactivity, The principal mechanism of brain damage in autism spectrum disorders and potentially HI is the deprivation from the glucose and oxygen psychotic symptoms such as schizophrenia. supply and also by initiating a cascade of Hypoxic-ischemic encephalopathy may be biochemical events leading to cell dysfunction and associated with multiorgan failure, including: renal finally to cell death, it consists of two phases: failure, hepatic failure, hematologic abnormalities, phase 1, consists of an early energetic failure such as polycythemia, hyperviscosity, which occur at the cellular level because of loss of disseminated intravascular coagulation, cardiac oxygen to the brain with a rapid cytotoxic edema dysfunction, and gastrointestinal dysfunction and necrotic cell death (Loverro et al., 2017). This (Wachtel and Hendricks, 2011). is followed by latent period (therapeutic window) Laboratory investigations should include the which occurred within 6 hours of insult. During this following changes: umbilical artery blood gas, reperfusion period, many enzymes such as metabolic acidosis, pH < 7 or base deficit ≥ 12 cyclooxygenase, xanthine oxidase, and mmol/L consistent with intrapartum hypoxia lipoxygenase can increase the production of free (Carrascosa et al., 2014). Serum electrolyte levels radicals and NO. That results in lipid peroxidation are markedly low as serum sodium, potassium, and damage of DNA and proteins and later and chloride levels and accompanied by presence apoptosis and damage to the brain of the newborn of reduced urine flow with extreme weight gain occur (Cross et al., 2010)& (Pacher et al., 2007). might indicate acute tubular damage or During this period an intervention might be development of syndrome of inappropriate effective in reducing the severity of the ultimate antidiuretic hormone (SIADH) secretion which brain damage., then phase 2 of cell death, a late occurs particularly in the initial 2-3 days of life energetic failure, which occurs usually during (Banerjee et al., 2013). reperfusion and reoxygenation hours after the Imaging studies as Cranial Ultrasonography is initial event and mostly lasts for days (Volpe, used to exclude structural abnormalities and 2008). The mechanisms of inflammation and detect cysts, calcifications, cerebral hemorrhage epigenetic changes may lead to an impairment or or atrophy and detect cortex and basal ganglia alteration of axonal growth, synaptogenesis and lesions. However, CS cannot allow early neurogenesis (Dixon et al., 2015). identification of asphyxial brain injury that may The etiology may be due to maternal causes develops evident between 24 and 72 hours after as Inadequate oxygenation of maternal blood, birth (Massaro et al., 2012). Head CT scanning is Low maternal blood pressure, Inadequate not a sensitive modality in evaluation of HIE. That relaxation of the uterus to permit placental filling, is because of the high water the neonatal brain Premature separation of the placenta, impedance and high protein in the (Pearce to the circulation of blood through the umbilical et al., 2012). Magnetic Resonance Imaging (MRI) cord, placental insufficiency from toxemia or post- is an ideal imaging modality in early evaluation of maturity or fetal causes as failure of oxygenation, brain injury in asphyxiated term neonates severe anemia and Severe shock. Neurological (Kudrevičienė et al.,2013). Near-Infrared dysfunction in form of neonatal encephalopathy Spectroscopy (NIRS) is considered as a non- which may present as: subnormal or depressed invasive tool for evaluating cerebral level of consciousness, respiratory depression, hemodynamics and evaluating oxygenation abnormal muscle tone and strength and seizures (Enns,2005). Other investigations as (Douglas and Weiss, 2015). (EEG) Used to recognize The complications with HIE may include: and monitor for seizures, on the other hand, neurological complications as multiple or severe normal EEG findings may not necessarily mean neurodevelopmental disabilities, Seizures, that the brain is healthy (Spitzmiller et al., 2007). Hearing loss (especially in infants with other New markers of tissue injury prepared as

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Sayed et al., Serum GFAP and CT in Neonates with Hypoxic Ischemic Encephalopathy. indicators of hypoxic-ischemic injury and as (GFAP) in neonate with HIE and its relation to possible predictors of outcome in prinatal grading. This study also had identified the role of asphyxia include Plasma Hypoxanthine cranial computed tomography in diagnosis of Vasopressin and Erythropoietin (Szczapa et al., hypoxic ischemic encephalopathy and its relation 2002), (GFAP) (Blennow et al., 1995), Nitric Oxide to GFAB finding. level increases in CSF with the severity of HIE between 24-72h following asphyxia (Ergenekon et MATERIALS AND METHODS al.,1999), elevated Aspartate, Glutamate and This study is a case-control study was carried Taurine in CSF (Gucuyener et al., 1999), Urinary out on 50 neonates babies , they were selected Lactate, (Huang et al., 1999), Protein S-100 and from (NICU) of Rashid General Hospital in CK-BB (Brain Specific Creatine Kinase) Alexandria, Egypt during the period from March (Nagdyman et al., 2001), Longitudinal S-100B 2015 to November 2017 defined according to the protein in urine soon after birth (Gazzolo et al., following criteria, it includes two groups: Group 2004) and elevation of cord Troponin (cTn I) (1): The cases group: It is including 30 cases, with (Kadir et al., 2004). inclusion criteria are full term newborn babies with Astrocytes are one type of glial cells in the gestational age between 37 to 42 weeks and CNS, astroglia, are characterized by a unique perinatal asphyxia which defined as the presence structural protein, (GFAP) which presents in non- of at least two of the following conditions; Signs of myelinating Schwann cells inside the PNS and fetal distress (heart rate of less than 100 beats per also in the enteric glia cells which is considered a minute, late decelerations, or an absence of heart part of the enteric (ENS) rate variability), thick, meconium stained amniotic (Gulbransen and Sharkey, 2012). It is responsible fluid and respiratory depression, hypotonia, or for the cytoskeleton structure of glia cells, bradycardia, APGAR score of 4 or less at one maintaining their mechanical strength, supporting minute or 6 or less at five minutes, a need for neighboring neurons and the blood brain barrier resuscitation for more than 1 minute with positive (BBB), playing a role in mitosis by adjusting the pressure ventilation and oxygen immediately after filament network present in the cell (Venkatesh et birth or blood pH value of less than 7.20 or a base al., 2013) and in repair after CNS injury (Cullen et deficit of at least 12 mmol/L within the first hour al., 2007). It is increased also in Alexander after birth. Group (2) The control group: disease, (Chen et al., 2011). The expression of Apparently healthy (20) newborns of matched some GFAP isoforms usually decrease in gestational age, birth weight and sex, free of any response to acute infection or of the above inclusion criteria and an uneventful as Wernicke's Encephalopathy, chronic infection postnatal clinical course during first week. with HIV-1, varicella zoster, pseudorabies, Down's All Neonates were subjected to the following: syndrome, schizophrenia, bipolar disorder and depression (Middeldorp and Hol, 2011). Full history taking: Mechanism of GFAP as biomarker protein for As gestational age, prenatal history for pre- acute CNS injury in hypoxic ischemic existing maternal or fetal problems and natal encephalopathy: The overall concept is that brain history suggestive of perinatal asphyxia as injury may cause release of (GFAP) and (GFAP- abnormal uterine contractions, prolonged labor, BDP) and from injured astrocytes directly to the mode of delivery, analgesia, anesthesia, amniotic interstitial fluid (ISF) / extracellular fluid, later fluid (normal, offensive or meconium stained), release to the circulating blood either by the direct resuscitation by oxygen, Ambu bag, endotracheal venous drainage or by diffusing and passing BBB intubation, chest compressions, medications, which becomes permeable (Plog, 2015). APGAR score at 1, 5 and 10 minutes (Apgar, A previous study demonstrated the correlation 1953) and postnatal history: for pulmonary, between GFAP level and MRI injury at 12 and 24 cardiovascular or neurological abnormalities. hours of life. Taken together, the results suggested that GFAP considered as an important 2- Thorough clinical examination: biomarker in the prediction of regions in brain Including birth weight, length and head injury in neonates with HIE during the first 24 circumference, neurological examination hours of life (Okonkwo, 2013). including: level of consciousness, neonatal reflexes as suckling and Moro, presence or Aim of the Work absence of seizures and Sarnat and Sarnat It was the assessment of the level of serum staging and according to the criteria of Sarnat,

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HIE was classified as mild (Grade 1) if hyper- 4-Imaging excitability, hyper-alertness, or hyper-reflexia Cranial CT which was carried on the fourth persisted without seizures for at least 24 hours day 4 postnatal. CT scans are reviewed from the after birth; as moderate if the infant was lethargic, caudal to cephalic levels. The scans are obtained had hypotonia, weak primitive reflexes, pupil at a 15- to 20-degree angulation to the miosis, and seizures; and as severe if the infant canthomeatal line. had apnea, flaccid weakness, frequent seizures, decelerated posture, or coma (Sarnat and Sarnat, Ethical Considerations: 1976) and Chest, heart and abdominal An informed written consent from all parents of examinations cases and control groups before getting them involved in the study according to the standards of 3. Laboratory Investigations including: the Medical Research Ethics Committee of the Cord blood sample for blood gas immediately National Research Centre, approval number: within hour after birth, Complete blood count 16/109. The steps of the study, the aim, the (CBC), C - Reactive Protein, Serum electrolytes: potential benefits and hazards, all were discussed e.g. Na, K and Ca, Serum urea and creatinine, with the parents of the cases and control groups ALT and AST and Random blood sugar (RBS). and Confidentiality of all data was ensured. For GFAP, two samples, 1st Sample at 0-6 hours and 2nd sample at 48-72 hours postnatal for RESULTS cases group and one sample to control group 1st Comparison between the two studied groups day postnatal. Sample collection: Venous blood regarding sex, gestational age and weight shows samples (EDTA and Serum) about 2-4 cm were that there was no statistically significant difference taken serially from each patient at the first 6 hours between cases and control regarding sex, post-natal then at 2rd to 3rd day after birth and gestational age and weight (p value > 0.05) and from the controller within the first hours postnatal. also no statistically significant difference regarding For preparation of serum, samples were allowed maternal data (age, parity and mode of delivery) to clot upright at room temperature for 30 min in between the two studied groups (70% of the processing laboratory before centrifugation for 15 mothers of cases group had obstructed labor and minutes at approximately 1000 x g at room 37% of them had medical disease). It shows temperature, then separated and immediately highly statistically significant difference between frozen and stored at -20°C or -80°C until the time the two studied groups regarding APGAR at 5 of analysis. All the laboratory tests are done in minutes, muscle tone, Moro reflex and the Immunogenetics Lab., National Research Centre, presence of respiratory distress (p value <0.005). Dokki, Giza, Egypt. Comparison between the two studied groups GFAP level was measured in samples using the regarding ABG parameters (pH, PaCO2, PaO2, Quantitative Sandwich Enzyme Immunoassay HCO3) shows highly statistically significant technique. Antibody specific for GFAP has been difference in parameters of ABG (pH, PaCO2, pre-coated onto a microplate. Standards and PaO2, HCO3) in the cases group compared to the samples were pipetted into the wells and any control group (p value <0.005). Comparison GFAP present was bound by the immobilized between the two studied groups regarding CBC antibody. After removing any unbound shows highly statistically significant decrease in substances, a biotinylated detection antibodies platelet counts in the cases group compared to specific for Human GFAP and Avidin- Horseradish the control group (p value <0.005) and no Peroxidase (HRP) conjugate were added to each statistically significant difference regarding well and incubated. Then, free components were hemoglobin level and white blood cells between washed away and the substrate reagent was the two studied groups (p value >0.05). added to each well, only those wells that contain Comparison between the two studied groups Human GFAP, biotinylated detection antibody and regarding the investigations of all cases shows Avidin-HRP conjugate gave blue color. The O.D. highly statistically significant difference regarding was measured by spectrophotometer at wave urea, creatinine, serum sodium and liver function length 450 nm, it is proportional to the between the two studied groups (p value <0.005) concentration of Human GFAP, finally the and no statistically significant difference between concentration of Human GFAP was calculated by the cases group and the control group regarding comparing the O.D. of the samples with a CRP, RBS and serum potassium (p value > 0.05). standard curve (Mondello, et al., 2010).

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Table (1): Comparison between the two studied groups regarding neonatal examinations at birth.

It shows highly statistically significant difference between the two studied groups regarding APGAR at 5 minutes, muscle tone, Moro reflex and the presence of respiratory distress (p value <0.005). Table (2): Comparison between the two studied groups regarding GFAP at 1st sample (0-6 hours) and 2nd sample (48-72 hours postnatal)

It shows statistically significant difference regarding GFAP at 2nd sample between the two studied groups (p<0.05) and no statistically significant difference between the cases group and the control group regarding GFAP at 1st sample (p> 0.05).

Table (3): Comparison between serum (GFAP) at 1st&2nd sample in the cases group

It shows highly statistically significant increase in serum GFAP at 2nd sample than 1st sample in the cases group (p<0.005).

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Table (4): Correlation of GFAP at 2nd sample with the Sarnat staging at the time of 2nd sample in the cases group.

It shows positive correlation between the GFAP at 2nd sample and Sarnat staging time of 2nd sample.

Figure (1): Shows positive correlation between the brain parenchyma and the staging of the cases group

Figure (2): shows correlation between the ventricular size and GFAP at 2nd sample in the cases group.

Figure (3): Shows the correlation between the presence of brain edema and GFAP at 2nd Sample in the cases group.

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Table (5): Correlation between the CT finding and the Sarnat staging of the cases group

It shows positive correlation between CT finding and staging of HIE in the cases group

Table (6): Correlation of GFAP at 2nd sample with the CT finding in the cases group.

One Way ANOVA ; *: Independent t-test

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NS: Nonsignificant; S: Significant; HS: Highly significant

Table (7): Shows cut off point more than0.655 between cases and control and AUC (Area Under the Curve) 0.995 regarding GFAP at the 2nd sample.

Figure (4): Shows ROC curve regarding serum GFAP at 2nd sample in the cases group and the control group.

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Figure (5): Shows example of our CT findings in cases group showing loss of differentiation between gray and white matter and constricted ventricle correlation between GFAP at 2nd sample and CT The percent of the cases group using mechanical finding in the cases group. ventilation was 56.7% and about 33.3% of the Sarnat staging of the cases group at the time cases died and 66.7% were discharged. of 2nd sample shows that stage 1 was high Comparison between the three stages of the percentage (40%) more than 2 and 3 (33.3%& cases group regarding all investigation and ABG 26.7%) respectively. shows highly statistically significant difference Correlation of GFAP at 1st sample and 2nd between the three stages of the cases group sample with the Sarnat staging at the time of 2nd regarding urea, creatinine (p value<0.005) and sample in the cases group shows positive significant difference in pH (p value <0.05), while correlation between the GFAP at 2nd sample and no significant difference regarding other Sarnat staging at the time of 2nd sample (p<0.05). investigations (p value >0.05). Comparison Correlation between GFAP at 1st and 2nd between the stage 1of the cases group and the sample and APGAR at 5,10 minutes and other control group regarding serum GFAP at 1ST investigations of the cases group shows sample and 2nd sample time shows highly statistically significant negative correlation statistically significant difference between the two between (hemoglobin and platelets) and 2nd previous studied groups regarding serum GFAP at sample of GFAP while there was significant 2nd sample (p<0.005), while no difference positive correlation between 1st and 2nd sample regarding GFAP at 1st sample (p>0.05). of GFAP and serum creatinine. Comparison between the stage 2 of the cases group and the control group regarding serum DISCUSSION GFAP at 1ST sample& 2nd sample time shows Hypoxic-ischemic (HI) brain injury is highly statistically significant difference between considered as one of the main causes of the two previous studied groups regarding serum disabilities in term-born neonates, also it is one of GFAP at 2nd sample (p value < 0.005), while no the most important causes of brain damage in the difference regarding GFAP at 1st sample (p value newborn period due to the deprivation of the > 0.05).Comparison between stage 3 of the cases neural tissue from oxygen and glucose (Felipe et group and the control group regarding serum al.,2013). Due to difficulties in diagnosis and GFAP at 1st sample and 2nd sample shows treatment of HI injury, we are in need to find statistically significant difference between the two accurate method to diagnosis, although many previous studied groups regarding serum GFAP at potential biomarkers of brain damage exist, serum 1st sample (p<0.05) and high statistically (GFAP) hold significant promise in this population. significant difference in 2nd sample (p>0.005). It It was measured in adult patients after , in shows positive correlation between the GFAP at cardiac arrest, or traumatic brain injury aiming to 2nd sample and Sarnat staging time of 2nd provide prognostic data on survival or residual sample. deficits. GFAP is a type III IF which forms a part of The (CT) finding among the cases group the cytoskeleton in the mature astrocytes, and shows that 26.7% of the cases group had consider a specific marker of differentiated hypoattenuation of gray matter and brain edema, astrocytes and an ideal blood marker for brain about 20% had compressed ventricle and 13.3% injury in neonates (Day and Thompson, 2009). had midline shift. Intervention is limited and mostly supportive at shows highly statistically significant negative this time, Cranial CT is commonly used for

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Sayed et al., Serum GFAP and CT in Neonates with Hypoxic Ischemic Encephalopathy. evaluation of term and near-term infants in insufficiency in addition to other factors as suspected brain injury cases, it performs relatively diabetes mellitus, rupture uterus, premature poorly in delineation of the common patterns of rupture of the membranes and history of the brain injury in neonatal encephalopathy, that obstructed labour. This result agrees with AZhar include deep nuclear gray matter and white matter et al., (2010) who reported the same results on injury (Martinez et al.,2011). the study done on 181 neonates with HIE. The cases group represented both gender: There was no significant difference between male 60% and female 40% with mean gestational the two studied groups regarding the mode of age (37.67±1.32) weeks and mean birth weight delivery (CS and VD) with p value (0.064). This (2.93±0.47) kg and control group represented result agrees with Bibi, (2012) who reported the both gender male 50% and female 50% with same results. mean gestational age (37.85±0.93) weeks and In our study we evaluated the cases group mean birth weight (3.15±0.35) kg. clinically two times, 1st time soon after birth and In our study, no statistically significant 2nd time 48-72 hours after birth, we found with the difference was found between the two studied 1st evaluation 70% of the cases group had groups regarding sex, gestational age and birth disturbed level of consciousness and 80% of them weight with p value (0.485, 0.594& 0.086) had convulsion, also there was highly statistically respectively and this result agree with Hesham et significant difference in APGAR score at 5 minute, al., (2005) who studied 20 full term neonates with muscle tone, Moro reflex and respiratory distress HIE and found also no statistically significant between the cases group and controller with p differences with p value (0.16& 0.71) respectively value (0.000) for all (Table 1) and this result in for birth weight and GA, also agree with Seema et agreement with Douglas et al., (2014) who al., (2014) who found no difference between the reported the same results in the study done on full cases and control group regarding sex with p term neonate with HIE when examined at birth. value (0.1). In our study there was highly statistically Regarding the hypoxic group male affected significant difference in arterial blood gas finding 60% more than female 40%, but not significant done immediately within hour after birth (pH, difference. This greater proportion of males PCO2, PaO2, HCO3) between the cases group affected by encephalopathy has been also noted and the controller with (p=0.000) for all and this by other studies (Nadia and John, 2001). In result agree with Hesham et al., (2005) who addition to Nunez and McCarthy (2003) reported reported the same results. Boskabadi et al., that male gender is more sensitive to brain injury (2010) also found that neonates with HIE had a than females. Hafiz et al., (2014) in Pakistan picture of acidosis and hypoxia with low pH value found also that male was more prone to perinatal of less than 7.2 or a base deficit of at least 12 asphyxia. This may indicate that some cases of mmol/l within the first hour after birth. encephalopathy are genetic in origin or that males There was no significant difference between are more vulnerable to factors causing newborn the two studied groups regarding hemoglobin encephalopathy or may be due to differences in level and white blood cells with p value (0.06& myelination rates between male and female fetus 0.089) respectively but there was highly (Nadia and John, 2001). statistically significant decrease in platelets count Our study showed no significant difference with p value (0.000). This result agrees with Shah between the two studied groups regarding age of et al., (2004) who found that about (32-54%) of the mothers and parity with p value (0.457& neonates with HIE had hematological disturbance 0.400) respectively and this agree with Chalak, including thrombocytopenia, and coagulopathy. (2014) who found no relation between maternal Coagulation disturbances and thrombocytopenia variable like age and parity and HIE. that occurred after birth asphyxia have This study showed regarding the cases group multifactorial origin, as alterations in blood or that 37% of the mothers had medical disease oxygen supply to the liver and bone marrow may (20% mothers hypertensive, 13.5% diabetic, 3.5% negatively impact the synthesis of clotting factors had polyhydramnios) also regarding the history of and platelets, respectively. Disseminated presence obstructed labour (70% positive and Intravascular Coagulation (DIC) is frequently 30% negative). This result agrees with Bukowski described following asphyxia in the newborn et al., (2003) who reported that the major (Bauman et al.,2011). antenatal contributing factor leading to HIE is There was highly statistically significant maternal hypertension due to placental difference regarding urea and creatinine between

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Sayed et al., Serum GFAP and CT in Neonates with Hypoxic Ischemic Encephalopathy. the two studied groups with (p=0.000, 0.004) decrease in other investigation like (Hg, WBCs& respectively. This results in agreement with Platelets) in stage 3 more than stage 1&2 but not Hankins et al.,(2002) who reported that the kidney statistically significant. These results agree with injury may represent the best systemic marker of Beken et al., (2014) who reported that significant brain injury, Serum creatinine and blood urea worsening in the results of kidney function tests, concentrations reflect the glomerular damage that pH and lactate value as the stage of HIE tend to increase progressively and reach the peak progressed, also reported that no statistically in the days following the injury. significant difference was detected among the We found highly statistically significant three groups with regard to (Hb) and leukocyte increase in the cases group compared to the levels. However, platelet count was the lowest in control group regarding liver function (AST& ALT) the cases group with Stage III HIE in study done with (p=0.000) but there was no statistically on 94 neonates with HIE. difference regarding blood glucose level with In our study there was no significant (p=0.330) between the cases and the controller, difference between the cases group and control the same with (Efron et al., 2003) who found that regarding GFAP at 0-6 hours age with (p=0.267), liver dysfunction is mainly showed increased but there was statistically significant difference hepatocellular enzymes however more extensive between the level of GFAP level at 48-72 hours damage may develop. Fluctuations of blood age and the GFAP level in the control group with glucose concentration usually observed, with (p= 0.040) (Table 2),this results agree with hypoglycemia is most common. On the other Douglas et al., (2014) who reported that GFAP hand, hyperglycemia can also lead to or was not significantly elevated when compared to a aggravate brain damage. control group at 0-6 hours of age (p=0.7) but there Regarding to our study there was high was statistically significant increase regarding statistically significant decrease in serum Na in GFAP at 48 -72 hours age, also Massaro et al., cases group compared to control group with (2013) have recently reported optimal time of (p=0.001). These results agree with Basu et al., detection GFAP at 24 and 72 hours of life in study (2010) who showed in their study significant done on neonates with HIE. hyponatremia (p < 0.001). Gupta et al., (2005) Table 3 shows highly statistically significant also observed that asphyxiated babies had lower difference between GFAP at 6 hours and 48-72 mean serum sodium levels as compared to the hours age regarding the cases group with control group (p <0.001). So daily assessment of (p=0.003), this result agrees with Ennen et al., serum electrolytes is valuable till the infant's (2011) who reported serial increase in GFAP from status improves. Low serum sodium with reduced birth to 96 hours age. urine flow and increased weight gain can indicate In the present study we found 26.7%of the acute tubular damage or develop syndrome of cases group had a picture of brain edema like inappropriate antidiuretic hormone (SIADH) hypoattenuation of gray matter, compressed secretion, particularly during the initial 2-3 days of ventricle and midline shift. The correlation life (Banerjee et al., 2013). There was no between the CT finding and the Sarnat staging of statistically significant difference between the the cases group shows positive correlation cases group and the control group regarding between CT finding and staging of HIE in the serum potassium (p value > 0.05) on the other cases group (Table 5) & (Figure 1). Our results hand, (Banerjee et al., 2013) noticed marked agree with Justin (2014) who found that 20% of decrease in serum sodium, potassium, and HIE group had brain edema in his study. chloride levels in those patients. HIE is associated with that In the present study we classified the cases reduces the attenuation of gray matter on group into 3 stages by Sarnat staging system two unenhanced CT images and results in loss of times, 1st time we found that stage 2 are more distinction between gray matter and white matter than other stages, they were 13 (43.3%) neonates while, in severe HIE, there may be inversion of and the 2nd time we found that stage 1 more than normal attenuation relationship between the gray other stages (40%). and white matter (a phenomenon known as the We found highly significant increase between reversal sign) (Gentsch et al., 2015). variable like urea and creatinine with increase The present study showed statistically staging of HIE in the cases group with p value significant increase in the level of GFAP at (6 (0.001& 0.002) respectively and significant hours and 48-72hours) with the stages 2&3 of the increase in pH (p value 0.014), while there was cases group compared with stage 1, this result

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Sayed et al., Serum GFAP and CT in Neonates with Hypoxic Ischemic Encephalopathy. agrees with Chalak, (2014) who reported that between the case and the control group regarding GFAP levels can persist significantly higher in 2nd sample of GFAP was more than 0.655 with neonates have moderate to severe HIE when AUC (area under the curve ) was 0.995 (Figure compared with those have mild HIE with (p=0.02), 4)& (Table 7) compared with the study done by and this confirmed the positive relationship Douglas et al., (2014) who also agree with us and between serum level at birth and degree of found cut off point was more than 0.64 and AUC encephalopathy. was 0.95, also agree with Chalak, (2014) who In our study there was statistically significant found AUC 0.85, cut off point was more than 0.55 correlation between increase the level of GFAP at and positive predictive value 100%,this means 48-72 hours age and CT finding mainly brain that the diagnostic accuracy of GFAP is edema (p=0.008) (Table 6) & (Figures 2&3). statistically significant in discriminating between On reviewing literatures no previous studies cases and control, accuracy was 99.5%. were found the relation of serum GFAP and CT finding. All studies were found conducted on adult. CONCLUSION Mondello et al.,(2011) reported in his study done We concluded that Levels of (GFAP) are on adult with brain injury that relationships higher in cases of HIE than control group and this between structural changes detected by CT and increase correlated with the severity of the biomarker like GFAP which may reflect a different disease. There is Serial increase in level GFAP injury pattern and provide better characterization from birth to 48-72 hours postnatal in HIE of subjects at risk for specific types of cellular neonates and finally, Brain edema is a common damage than that obtained with neuroimaging finding in cranial CT in cases of HIE and this alone, as well as provide valuable information finding correlated with the severity of the disease about injury severity and outcome. and with the level of GFAP. In our study there was statistically negative correlation between (Hb, platelets) and GFAP at Recommendations 48-72 hours age with p value (0.023& 0.006 Proper antenatal care should be available for respectively), while there was statistically positive all mothers with early identification and proper correlation between GFAP at (0-6& 48-72) hours management of high risk pregnancies (e.g. age and serum creatinine with p value (0.012& Diabetes, hypertension, etc), special care for 0.024) respectively. These results agree with delivery and NICU for early observation should be Chalak (2014) who reported that GFAP is the only made available to at risk neonates, large sample serum biomarker which correlates significantly study to measure serum GFAP in HIE within 6 with multiple organ dysfunctions: Hb level (p= hours postnatal to early identify neonates with 0.01), platelets (p= 0.02), and creatinine (p= 0 .0) poor prognosis and further assessment of serum on a study was done on neonates with HIE. GFAP in HIE neonates undergoing to Our study showed statistically significant pharmacological neuroprotective measurements correlation between n the GFAP at (48-72hours) as EPO, Barbiturate and Magnesium Sulphate. and Sarnat staging at the time of 2nd sample with p value (0.025), it means that GFAP can be used CONFLICT OF INTEREST as a prognostic marker (Table 4). This result The present study was performed in absence of any agrees with Ennen et al., (2011) who reported in a conflict of interest. study done on 46 neonates with HIE that GFAP serve as surrogates of disease injury, evolution, ACKNOWLEGEMENT and outcome, GFAP levels were predictive of an We thank the National Research Centre, abnormal magnetic resonance imaging film and Dokki, Giza for supporting this work and Rashid correlated with short-term functional outcomes, General Hospital in Alexandria for their help and additionally Pelinka et al., (2004) reported that assistance. GFAP was correlated with poor outcomes in adult patients after stroke, in cardiac arrest, or in AUTHOR CONTRIBUTIONS traumatic brain injury, also Lumpkins et al., (2008) SKS, HGE, NAEA and NMK reviewed the and Kaneko et al., (2009) reported that GFAP has manuscript, NMK performed the laboratory tests, is used as a predictor of poor neurological THNM revised the radiological examinations’ outcomes for children requiring extracorporeal reports. GFE collected samples, performed the membrane oxygenation. Finally we found in our study cut off point

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