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Glial fibrillary acidic protein as a biomarker for periventricular white matter injury

Published:March 04, 2013DOI:https://doi.org/10.1016/j.ajog.2013.02.049

      Objective

      Periventricular white matter injury (PWMI), a precursor of cerebral palsy, traditionally is not diagnosed until 6 weeks of life by head ultrasound scanning. We sought to determine whether early neonatal glial fibrillary acidic protein (GFAP) levels could identify PWMI in low birthweight (<2500 g) infants.

      Study Design

      Each case with PWMI on head ultrasound scanning at 6 weeks of life from April 2009 to April 2011 was matched by gestational age and mode of delivery to 2 subsequent neonates with a normal head ultrasound scan. GFAP was measured in cord blood at birth, at neonatal intensive care unit admission, and on days 1-4 of life.

      Results

      During this 2-year period, 21 cases with PWMI with gestational age 27.4 ± 3.3 weeks were compared with 42 control infants. The incidence of cesarean delivery was 61.9% in both groups. GFAP was not significantly different in cord blood or at neonatal intensive care unit admission but was significantly elevated on day 1 (median, 5-95%; 0, 0-0.98 ng/mL cases; 0, 0-0.06 ng/mL control infants; P = .03), day 2 (0, 0-1.21 ng/mL; 0, 0-0.05 ng/mL, respectively; P = .02), day 3 (0.05, 0-0.33 ng/mL; 0, 0-0.04 ng/mL, respectively; P = .004), and day 4 (0.02, 0-1.03 ng/mL; 0, 0-0.05 ng/mL, respectively; P < .001). The odds of the development of PWMI significantly increased with increasing levels of GFAP from day 1-4 of life when adjustment was made for preeclampsia, antenatal steroid administration, and neonatal chronic lung disease.

      Conclusion

      The ability to predict PWMI with a blood test for GFAP shortly after birth opens the possibility for rapid identification of infants for early intervention and provides a benchmark for the qualification of new therapies to improve neurodevelopmental outcomes.

      Key words

      For Editors' Commentary, see Contents
      As a consequence of preterm birth, low birthweight (LBW; <2500 g) infants are at increased risk for a spectrum of cerebral white matter abnormalities termed periventricular white matter injury (PWMI). Head ultrasound imaging routinely is performed in premature neonates in the first week of life to rule out intraventricular hemorrhage (IVH) and at 6 weeks for identification of PWMI. The incidence of PWMI is higher in infants who sustain IVH possibly because the hemorrhage provides a rich source of iron for the generation of reactive oxygen species that leads to oligodendrocyte death.
      • Volpe J.J.
      Neurobiology of periventricular leukomalacia in the premature infant.
      When PWMI is present, it is associated with cerebral palsy in 60-100% of survivors.
      • Leviton A.
      • Paneth N.
      White matter damage in preterm newborns: an epidemiologic perspective.
      Cerebral palsy affects 6-9% of infants who are born at <32 weeks’ gestation and as many as 28% of infants who are born at <26 weeks’ gestation.
      • Milligan D.W.
      Outcomes of children born very preterm in Europe.
      The fetal transition at birth is known to be a particularly vulnerable time for the preterm infant brain; however, we lack the means to identify infants objectively who are at risk for long-term neurologic injury in the early neonatal period. Currently available tools to identify the neonate who is at risk for encephalopathy shortly after birth include fetal heart rate tracings, Apgar scores, umbilical cord gases, and physical examination of the newborn infant, all of which lack precision.
      In adult brain injury, such as traumatic brain injury and stroke, the measurement of circulating brain proteins have demonstrated significant diagnostic and prognostic potential.
      • Wunderlich M.T.
      • Wallesch C.W.
      • Goertler M.
      Release of glial fibrillary acidic protein is related to the neurovascular status in acute ischemic stroke.
      • Vos P.E.
      • Lamers K.J.
      • Hendriks J.C.
      • et al.
      Glial and neuronal proteins in serum predict outcome after severe traumatic brain injury.
      • Lumpkins K.M.
      • Bochicchio G.V.
      • Keledjian K.
      • Simard J.M.
      • McCunn M.
      • Scalea T.
      Glial fibrillary acidic protein is highly correlated with brain injury.
      • Kaneko T.
      • Kasaoka S.
      • Miyauchi T.
      • et al.
      Serum glial fibrillary acidic protein as a predictive biomarker of neurological outcome after cardiac arrest.
      A number of proteins, which includes the highly brain-specific glial fibrillary acidic protein (GFAP), have been used in adults and children to identify patients with stroke or traumatic brain injury in an effort to provide prognostic data on survival or density of residual deficits.
      • Vos P.E.
      • Lamers K.J.
      • Hendriks J.C.
      • et al.
      Glial and neuronal proteins in serum predict outcome after severe traumatic brain injury.
      GFAP is a brain-specific cytoskeletal intermediate filament protein that is found in the astroglia of the central nervous system and is a specific marker of differentiated astrocytes. Serum GFAP is derived entirely from brain and not secreted routinely in blood but is released only after cell injury or death. In adult patients who had sustained mild traumatic brain injury, a relation was found between GFAP measured directly after admission, imaging studies, and outcome determined by return to work.
      • Metting Z.
      • Wilczak N.
      • Rodiger L.A.
      • Schaaf J.M.
      • van der Naalt J.
      GFAP and S100B in the acute phase of mild traumatic brain injury.
      Our group has found that elevated serum GFAP levels in neonates who underwent extracorporeal membrane oxygenation were associated with acute brain injury and death.
      • Bembea M.M.
      • Savage W.J.
      • Strouse J.J.
      • et al.
      Glial fibrillary acidic protein as a brain injury biomarker in children undergoing extracorporeal membrane oxygenation.
      We have also found that, in term and near-term neonates with hypoxic-ischemic encephalopathy who were treated with whole body cooling, elevated GFAP levels on admission to the neonatal intensive care unit (NICU) and through day 4 of life were predictive of brain magnetic resonance imaging (MRI) abnormalities at 7 days of life and abnormal neurodevelopmental outcomes.
      • Ennen C.S.
      • Huisman T.A.
      • Savage W.J.
      • et al.
      Glial fibrillary acidic protein as a biomarker for neonatal hypoxic-ischemic encephalopathy treated with whole-body cooling.
      Our aim in this study was to determine whether GFAP levels that were measured within the first 4 days of life could be used to identify LBW neonates who are at risk for the development of PWMI.

      Materials and Methods

      This was an institutional review board–approved case control study of all liveborn, nonanomalous LBW (<2500 g) neonates who were born at a single university hospital and who were admitted to the NICU. Neonates with major congenital malformations, chromosomal abnormalities, or genetic syndromes were excluded. For GFAP studies, cord blood and the residual unused portion of serum from daily routine clinical laboratory tests were used. We collected cord blood at delivery and the remaining portion of serum from daily neonatal blood draws during the first 4 days of life for all neonates who were admitted to our NICU weighing <1500 g and for those with birthweights of 1500-2500 g with suspected neurologic morbidity at birth, which included prolonged hypotonia or seizures. Most of these samples were obtained by heel stick, but some samples were obtained from umbilical arterial lines. Residual serum was aliquoted and stored at –80°C until assayed. After neonatal discharge, we reviewed the results of head ultrasound scans that were performed at 6 weeks of life to identify infants with PWMI whose data were then compared with the subsequent 2 infants with normal head ultrasound scans at the same gestational age within 1 week and delivered by the same mode, matching in a 2:1 fashion. In addition, from this group, we compared those infants who experience the development of both IVH and PWMI with those infants with IVH only.
      Our GFAP assay has been described previously.
      • Bembea M.M.
      • Savage W.J.
      • Strouse J.J.
      • et al.
      Glial fibrillary acidic protein as a brain injury biomarker in children undergoing extracorporeal membrane oxygenation.
      • Ennen C.S.
      • Huisman T.A.
      • Savage W.J.
      • et al.
      Glial fibrillary acidic protein as a biomarker for neonatal hypoxic-ischemic encephalopathy treated with whole-body cooling.
      Case and control samples that were blinded to the neonatal head ultrasound imaging results were assayed at the same time. Samples and standards were assayed in duplicate. Assays were analyzed on a Sector Imager 2400 (Meso Scale Discovery, Rockville, MD) according to the manufacturer’s protocol. Values were reported as the mean concentration of a single sample that was assayed in duplicate. GFAP concentrations are a continual range from the lower to the upper limit of quantification for the assay (0.04–40 ng/mL).
      • Bembea M.M.
      • Savage W.J.
      • Strouse J.J.
      • et al.
      Glial fibrillary acidic protein as a brain injury biomarker in children undergoing extracorporeal membrane oxygenation.
      GFAP values <0.04 ng/mL, which is the lower limit of quantitation, were reported as 0. The GFAP assay has an interassay coefficient of variability of <2.5%. The intraassay coefficient of variability ranged from 0.18–3.07%. GFAP values that were obtained from plasma and serum were equivalent.
      All neonates had a head ultrasound scan within 1 week and at 2 weeks to rule out IVH and a third ultrasound scan at 6 weeks to rule out PWMI, as per our standard clinical practice. Head ultrasound scans were all performed in the NICU. Transfontanellar head ultrasonography was performed with state-of-the-art ultrasound equipment (Zonare Medical Systems, Mountain View, CA). Standardized optimized sets of coronal and sagittal images were obtained through the anterior fontanel with the use of curved and linear array transducers (8-17 MHz). Head ultrasound scans were evaluated by 2 pediatric neuroradiologists (A.T., T.A.G.M.H.) for overall white matter echointensity, gray-white matter differentiation, echointensity of the central gray matter, ventricular size (based on the largest ventricle size), germinal matrix hemorrhage (based on Papille’s classification from I-III),
      • Papile L.A.
      • Burstein J.
      • Burstein R.
      • Koffler H.
      Incidence and evolution of subependymal and intraventricular hemorrhage: a study of infants with birth weights less than 1,500 gm.
      periventricular hemorrhagic infarction (previously known as grade IV hemorrhage), and cystic periventricular leukomalacia. Diffuse white matter injury was defined as generalized white matter volume loss, with or without echo intensity alterations. Focal white matter injury was defined as focal areas of decreased echo intensity that represent remote ischemia or hemorrhage.
      Neonates with PWMI were scheduled for a comprehensive neonatal neurodevelopmental examination after discharge from the NICU, as per our standard of care. This examination draws from the work of many researchers in the area and was reported previously.
      • Allen M.C.
      • Aucott S.
      • Cristofalo E.A.
      • Alexander G.R.
      • Donohue P.K.
      Extrauterine neuromaturation of low risk preterm infants.
      This examination is corrected for gestational age at birth and assessed the emergence of extremity flexor tone, axial (neck, trunk, shoulder, and hip) tone, deep tendon reflexes, pathologic reflexes, primitive reflexes, postural head control, and sensory responses. These examinations were conducted by a single examiner (M.C.A.) who is certified in pediatrics, neonatology, and neurodevelopmental disabilities. The diagnosis of cerebral palsy was based on a persistently abnormal neurologic examination (eg, spasticity and/or variable tone and/or persistent primitive and pathologic reflexes) and functional impairment (including abnormal quality of movement).
      • Allen M.C.
      • Alexander G.R.
      Using motor milestones as a multistep process to screen preterm infants for cerebral palsy.
      In the case group with abnormal head ultrasound scans, GFAP levels were compared between infants who had cerebral palsy and those who did not.
      Infant and maternal medical records were reviewed to identify relevant clinical data. Intrauterine growth restriction was defined as an estimated fetal weight <10th percentile.
      • Hadlock F.P.
      • Harrist R.B.
      • Martinez-Poyer J.
      In utero analysis of fetal growth: a sonographic weight standard.
      Oligohydramnios was defined as an amniotic fluid index <5.0 cm at the time of the admission at which the delivery occurred. Preeclampsia was defined as proteinuria, edema, and the presence of new onset hypertension. The clinical diagnosis of chorioamnionitis was made in the presence of maternal fever, with the presence of at least one other finding of fetal tachycardia, uterine tenderness, or purulent vaginal discharge. Patients who received a diagnosis of clinical chorioamnionitis were started immediately on intravenous ampicillin and gentamicin, if not allergic. All the placentas in the study were examined by an attending pathologist at our institution. Histologic chorioamnionitis was diagnosed when any polymorphonuclear leukocytes were seen in either the chorion or amnion or in significant amounts in the subchorionic space. Histologic funisitis was diagnosed when polymorphonuclear leukocytes were seen in the umbilical cord. The diagnosis of nonreassuring fetal heart rate tracing was made by the physician who attended the delivery before performing a cesarean delivery.
      All statistical analyses were conducted using STATA software (STATA Intercooled, version 12.0; STATA Corporation, College Station, TX). Comparisons of cases of PWMI and matched control infants on demographic and clinical characteristics were conducted to ensure the success of the matching and to identify differences between the 2 groups. Linear regression was used to determine the association between gestational age and GFAP levels in the control population. Multivariable conditional logistic regression was conducted to determine whether GFAP levels in the first 4 days of life predicted PWMI at 6 weeks with adjustment for differences between cases and control infants. Cases with both IVH and PWMI were compared with neonates with IVH only with the use of independent samples t tests, χ2 test, and the Wilcoxon rank-sum test, as appropriate. GFAP was compared within the PWMI cases based on the later diagnosis of cerebral palsy with the Wilcoxon rank-sum test. Statistical significance was determined by a confidence interval that did not include 1.0 and a probability value < .05.

      Results

      During the 2-year period from April 2009 to April 2011, there were 177 inborn very low birthweight (VLBW) neonates without major congenital malformations who were admitted to our NICU, of which 18 cases of PWMI were identified, for an incidence of 10.2%. There were 221 inborn, nonanomalous LBW neonates with birthweight 1500-2500 g who were admitted to the NICU during this period, of which 3 (1.4%) had PWMI. These 21 cases with PWMI were matched to 42 control infants with normal head ultrasound scans. The mean gestational age and standard deviation for cases and control infants were 27.4 ± 3.3 and 27.4 ± 3.2 weeks, respectively. The incidence of cesarean delivery was 61.9% in both groups. Comparisons between cases and matched control infants on maternal demographic and clinical characteristics identified a number of similarities and differences (Table 1). Similarities (ie, nonstatistical differences) between cases and matched control infants included the incidence of twin gestation, intrauterine growth restriction, and clinical and histologic chorioamnionitis. Cases were significantly less likely to have preeclampsia and to have received antenatal steroids before delivery compared with matched control infants.
      Table 1Univariate analysis comparing maternal variables
      VariableCases (n = 21)Control infants (n = 42)P value
      Maternal age, y
      Data are given as mean ± SD
      27.8 ± 7.228.1 ± 7.4.86
      Median parity, n11.65
      Race, n (%).83
       White6 (28.6)9 (21.4)
       Black13 (61.9)30 (71.4)
       Other2 (9.5)3 (7.1)
      Cesarean delivery, n (%)13 (61.9)26 (61.9)1.0
      Twins, n (%)4 (19.0)5 (11.9).49
      Preterm labor, n (%)14 (66.7)19 (45.2).16
      Preterm premature rupture of membranes, n (%)5 (23.8)14 (33.3).49
      Preeclampsia, n (%)2 (9.5)15 (35.7).03
      P < .05.
      Antenatal magnesium, n (%)8 (38.1)22 (52.4).29
      Intrauterine growth restriction, n (%)07 (16.7).09
      Oligohydramnios, n (%)1 (4.8)5 (11.9).24
      Steroid administration, n (%)12 (57.1)35 (83.3).03
      P < .05.
      Abruption, n (%)5 (23.8)4 (9.5).15
      Nonreassuring fetal heart rate tracing, n (%)6 (28.6)5 (11.9).11
      Clinical chorioamnionitis, n (%)1 (4.8)11 (26.2).08
      Histologic chorioamnionitis, n (%)3 (14.3)16 (38.1).053
      Histologic funisitis, n (%)2 (9.5)8 (19.0).21
      Histologic placental infarct, n (%)1 (4.8)5 (11.9).73
      Univariate analysis that compared maternal variables between cases with periventricular white matter injury on head ultrasound scan at 6 weeks of life and control infants with normal head ultrasound scans who were matched by gestational age.
      Stewart. GFAP and periventricular white matter injury. Am J Obstet Gynecol 2013.
      a Data are given as mean ± SD
      b P < .05.
      Neonatal characteristics that were often associated with prematurity were also compared between cases and matched control infants (Table 2). The cases and control infants were similar with regard to birthweight, Apgar scores, umbilical arterial cord gases, and culture positive sepsis. During the first 4 days of life, there was no difference in hypocarbia or hyperoxia. During their neonatal hospitalization, there was no difference in Indocin exposure or need for high-frequency ventilation. Cases with abnormal head ultrasound scans were significantly more likely to have IVH and seizures. Cases had a higher incidence of chronic lung disease compared with matched control infants.
      Table 2Univariate analysis comparing neonatal variables
      VariableCases (n = 21)Control infants (n = 42)P value
      Birthweight, g
      Data are given as mean ± SD
      1077 ± 5441015 ± 458.41
      Apgar score <7, n (%)
       1-minute20 (95.2)40 (95.2)1.0
       5-minute14 (66.7)17 (40.5).06
      Cord pH
      Data are given as mean ± SD
      7.22 ± 0.197.27 ± 0.12.36
      Cord base deficit, mmol/L
      Data are given as mean ± SD
      4.4 ± 6.72.4 ± 2.1.19
      Cord pH <7.0 & base deficit >12 mmol/L, n/N (%)3/17 (11.8)2/32 (6.3).41
      Lowest arterial pCO2, mm Hg
      Data are given as mean ± SD
      32 ± 732 ± 7.91
      Highest arterial pO2, mm Hg
      Data are given as mean ± SD
      105 ± 31100 ± 58.58
      Indocin exposure, n (%)6 (28.6)14 (33.3).33
      High frequency ventilation, n (%)14 (66.7)22 (52.4).11
      Neonatal length of stay, d
      Data are given as mean ± SD
      72.0 ± 39.158.3 ± 42.4.22
      Respiratory distress syndrome, n (%)19 (90.5)41 (97.6).26
      Chronic lung disease, n (%)15 (71.4)20 (47.6).04
      P < .05.
      Necrotizing enterocolitis, n (%)2 (9.5)6 (14.3).56
      Death, n (%)04 (9.5).29
      Intraventricular hemorrhage, n (%)19 (90.5)11 (26.2)< .001
      P < .05.
      Seizures, n (%)9 (42.9)0< .001
      P < .05.
      Positive blood culture, n (%)2 (9.5)6 (14.3).58
      Positive cerebrospinal fluid culture, n (%)2 (9.5)0.11
      Positive urine culture, n (%)2 (9.5)3 (7.1).76
      Univariate analysis that compared neonatal variables between cases with periventricular white matter injury on head ultrasound scan at 6 weeks of life and control infants with normal head ultrasound scans who were matched by gestational age.
      Stewart. GFAP and periventricular white matter injury. Am J Obstet Gynecol 2013.
      a Data are given as mean ± SD
      b P < .05.
      Serum GFAP levels were available for comparison at 111/126 (88.1%) and 212/252 (84.2%) of the desired time points for cases and controls, respectively. Missing data were due to an inability to obtain cord blood at delivery, insufficient serum after clinically indicated tests had been performed, or absence of blood being drawn on some days. GFAP was not significantly different in cord blood at birth (median, 5-95%; 0.06 and 0-0.7 ng/mL cases; 0.04 and 0-0.56 ng/mL control infants; P = .97) or at NICU admission (0 and 0-0.22 ng/mL; 0 and 0-0.37 ng/mL; P = .98) but was significantly increased on day 1 (0 and 0-0.98 ng/mL; 0 and 0-0.06 ng/mL; P = .03), day 2 (0 and 0-1.21 ng/mL; 0 and 0-0.05 ng/mL; P = .02), day 3 (0.05 and 0-0.33 ng/mL; 0 and 0-0.04 ng/mL; P = .004), and day 4 (0.02 and 0-1.03 ng/mL; 0 and 0-0.05 ng/mL; P < .001; Figure 1) Multivariable conditional logistic regression showed that the odds of the development of PWMI increased with increasing levels of GFAP from day 1 to day 4 of life with adjustment for preeclampsia, antenatal steroid administration, and neonatal chronic lung disease (Table 3). GFAP levels did not change significantly with gestational age in the control infants on day 1 (R = .31; P = .08), day 2 (R = 0.2; P = .24), day 3 (R = 0.1; P = .48), or day 4 (R = 0.19; P = .92). There was a significant increase in cases with detectable GFAP levels of >0.04 ng/mL on day 2 (8/20 cases [40%]; 2/37 control infants (5.4%); P = .001), day 3 (9/17 cases [52.9%]; 0/36 control infants; P < .0001), and day 4 (8/18 cases [44.4%]; 3/30 control infants [10%]; P = .006). The sensitivity of detectable GFAP levels (>0.04 ng/mL) in the identification of PWMI on a head ultrasound scan at 6 weeks of life was 52.4% on day 1, 55.0% on day 2, 64.7% on day 3, and 66.7% on day 4; specificity was 91.2% on day 1, 91.9% on day 2, 94.4% on day 3, and 86.7% on day 4.
      Figure thumbnail gr1
      Figure 1GFAP as a biomarker for PWMI in low birthweight neonates
      The graph shows a comparison of cases with PWMI and control infants with normal head ultrasound scans at 6 weeks of life.
      GFAP, glial fibrillary acidic protein; PWMI, periventricular white matter injury.
      Stewart. GFAP and periventricular white matter injury. Am J Obstet Gynecol 2013.
      Table 3Multivariable conditional logistic regression of GFAP levels to predict PWMI
      TimeOdds ratio95% CIP value
      Birth3.58−2.96 to 10.1.28
      Neonatal intensive care unit admission14.5−3.0 to 31.9.10
      Day
       132.22.97–61.4.03
      P < .05.
       227.84.74–50.8.02
      P < .05.
       346.614.5–78.7.004
      P < .05.
       4424324–524< .001
      P < .05.
      Multivariable conditional logistic regression that was adjusted for preeclampsia, antenatal steroid administration, and chronic lung disease to determine whether glial fibrillary acidic protein levels from birth, neonatal intensive care unit admission, and the first 4 days of life predict periventricular white matter injury on head ultrasound scanning at 6 weeks of life.
      CI, confidence interval; GFAP, glial fibrillary acidic protein; PWMI, periventricular white matter injury.
      Stewart. GFAP and periventricular white matter injury. Am J Obstet Gynecol 2013.
      a P < .05.
      These cases and control infants were all preterm neonates at <35 weeks’ gestation, none of which were candidates for treatment with whole body cooling, which is limited to those with suspected hypoxic-ischemic encephalopathy who are at >35 weeks’ gestation. Only 3 of 17 of the preterm cases (11.8%) in this study had an umbilical arterial gas at birth that was consistent with metabolic acidosis from intrapartum hypoxia-ischemia (pH <7.0; base deficit, >12 mmol/L). The PWMI that was seen in these preterm neonates on a head ultrasound scan at 6 weeks of life was not related to intrapartum hypoxia-ischemia in the other 88.2% of cases, because their cord gas was normal. We did not find a difference in GFAP levels within the case group when the 9 cases with seizures were compared with the 11 cases without seizures at any time point from birth to day 4 of life (data not shown).
      Among all neonates with IVH, those who later experienced PWMI (n = 20) were compared with those with IVH only (n = 29). Neonates with IVH and PWMI did not differ from those with IVH only in gestational age (26.6 ± 2.7 and 27.1 ± 2.8 weeks, respectively; P = .52), birthweight (960 ± 444 and 952 ± 366 g, respectively; P = .94) or incidence of cesarean delivery (60% and 48.3%, respectively; P = .42). There was no difference in preeclampsia, steroid administration, or chronic lung disease between the groups. Those infants with IVH and PWMI had a significantly higher incidence of seizures (45% and 6.9%, respectively; P = .004) and severe (III or IV) grade (85% and 17.2%, respectively; P < .0001). GFAP was significantly increased in those with IVH and PWMI on days of life 2-4 (P = .04, .02, and .02; Figure 2).
      Figure thumbnail gr2
      Figure 2GFAP as a biomarker for PWMI in low birthweight neonates with IVH
      The graph shows a comparison of cases with both PWMI and intraventricular hemorrhage and control infants with IVH only.
      GFAP, glial fibrillary acidic protein; IVH, intraventricular hemorrhage; PWMI, periventricular white matter injury.
      Stewart. GFAP and periventricular white matter injury. Am J Obstet Gynecol 2013.
      We were able to obtain neurodevelopmental follow-up data for 15 of 21 of the cases with PWMI (71.4%). These neurodevelopmental examinations were conducted at 14.4 ± 6.6 months postnatally (range, 7–27 months) and were corrected for gestational age at birth. Cerebral palsy was diagnosed in 12 of 15 of these infants (80%). GFAP for the 3 cases without cerebral palsy was 0 on days of life 1-4. The mean, 5% and 95% for GFAP levels for the 12 cases with cerebral palsy was 0 and 0-0.99 ng/mL on day 1 (P = .27); 0 and 0-0.57 ng/mL on day 2 (P = .65); 0 and 0-0.33 ng/mL on day 3 (P = .26), and 0 and 0-0.13 ng/mL on day 4 (P = .31).

      Comment

      Although we chose to study LBW infants because of the high incidence of abnormal neurodevelopment in this population, most of the neonates that we identified as having PWMI were VLBW, which is a population in which PWMI is the predominant form of structural neonatal brain damage and who have a 25-50% occurrence of neurodevelopmental deficits and a 5-10% occurrence of major motor deficits, such as cerebral palsy.
      • Volpe J.J.
      Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances.
      Presently, head ultrasound imaging at 6 weeks of age is the gold standard for the diagnosis of PWMI. MRI at term that is equivalent in very preterm infants has been shown to identify moderate-to-severe cerebral white matter abnormalities that strongly predict adverse neurodevelopmental outcome at 2 years of age
      • Woodward L.J.
      • Anderson P.J.
      • Austin N.C.
      • Howard K.
      • Inder T.E.
      Neonatal MRI to predict neurodevelopmental outcomes in preterm infants.
      ; however, at this time MRI is not standard for these infants. In the present study, we demonstrate that levels of circulating GFAP on days 1-4 of life are elevated significantly in preterm neonates who are at risk for the development of PWMI and that, even among those neonates with IVH, GFAP can identify which infants are at high risk for the later development of PWMI. This allows for the identification of these infants much sooner than by either head ultrasound scan or MRI.
      Our control infants were significantly more likely to have received antenatal steroids before delivery, which agrees with previous research that showed a decreased risk for PWMI in preterm neonates who received antenatal betamethasone
      • Baud O.
      • Foix-L’Helias L.
      • Kaminski M.
      • et al.
      Antenatal glucocorticoid treatment and cystic periventricular leukomalacia in very premature infants.
      and emphasizes the importance of steroid administration in preterm neonates who are at risk for delivery. Neonates who receive antenatal steroids may have improved pulmonary function and be at lower risk for hypoxia, which could decrease their risk for brain injury. Cases with PWMI were also significantly more likely to have chronic lung disease, which is a known risk factor for neurologic impairment in VLBW infants.
      • Wilson-Costello D.
      Risk factors for neurologic impairment among very low-birth-weight infants.
      Neonatal chronic lung disease could be a source of hypoxia that could lead to brain injury. Control infants were significantly more likely to have been delivered from mothers with preeclampsia, and there is evidence that shows maternal preeclampsia may provide neonatal neuroprotection.
      • Wilson-Costello D.
      Risk factors for neurologic impairment among very low-birth-weight infants.
      Intravenous magnesium sulfate routinely is given to mothers with preeclampsia to prevent seizures; 3 large randomized placebo controlled trials found that antenatal magnesium sulfate therapy reduced the risk of cerebral palsy in children who survived very early preterm birth.
      • Crowther C.A.
      • Hiller J.E.
      • Doyle L.W.
      • Haslam R.R.
      Effect of magnesium sulfate given for neuroprotection before preterm birth: a randomized controlled trial.
      • Rouse D.J.
      • Hirtz D.G.
      • Thom E.
      • et al.
      A randomized, controlled trial of magnesium sulfate for the prevention of cerebral palsy.
      • Marret S.
      • Marpeau L.
      • Follet-Bouhamed C.
      • et al.
      Effect of magnesium sulphate on mortality and neurologic morbidity of the very-preterm newborn (of less than 33 weeks) with two-year neurological outcome: results of the prospective PREMAG trial.
      There was no difference in magnesium exposure between the groups in our study because it has become standard practice in our center to administer intravenous magnesium sulfate for neuroprotection to any mother who is at risk for delivering at <32 weeks’ gestation.
      Many new therapies for PWMI in the premature neonate currently are being investigated, which provides the impetus to identify neonates who are at risk in the early postnatal period. An example is erythropoietin and its derivative carbamylated erythropoietin that have been shown to decrease the risk of cerebral white matter injury in mouse models of PWMI.
      • Liu W.
      • Shen Y.
      • Plane J.M.
      • Pleasure D.E.
      • Deng W.
      Neuroprotective potential of erythropoietin and its derivative carbamylated erythropoietin in periventricular leukomalacia.
      Stem cell treatments have been studied in animal models of cerebral palsy and have been shown to produce beneficial effects.
      • Titomanlio L.
      • Kavelaars A.
      • Dalous J.
      • et al.
      Stem cell therapy for neonatal brain injury: perspectives and challenges.
      Specific brain biomarkers (such as GFAP) could play an important role as early surrogate outcome measures and benchmarks for the efficacy of these interventions.
      GFAP was not elevated significantly in the cord blood at birth or on NICU admission in these preterm neonates who were later diagnosed with PWMI, which suggests that brain injury was postnatal. Prenatally, the fetus could be protected by having adequate cerebral blood flow in utero because of placental perfusion. Not until after delivery when the neonates must maintain their own cerebral autoregulation do hypotensive episodes occur that lead to astrocyte injury; manifestations of this are not detectable by serum GFAP until the first day after birth. The first few days after birth are a critical period during which there may also be differences in ventilator support, maximal mean airway pressures, hyperoxia, hypocarbia, and administered drugs, which could contribute to these injuries. No such differences were found between cases and control infants in this study.
      Because of very weak cerebrovascular autoregulation, cerebral perfusion in preterm infants is dependent on systemic blood pressure
      • Khwaja O.
      • Volpe J.J.
      Pathogenesis of cerebral white matter injury of prematurity.
      ; pressure passivity increases with increasing degrees of prematurity in critically ill preterm neonates.
      • Gilmore M.M.
      • Stone B.S.
      • Shepard J.A.
      • Czosnyka M.
      • Easley R.B.
      • Brady K.M.
      Relationship between cerebrovascular dysautoregulation and arterial blood pressure in the premature infant.
      Blood pressure treatment in preterm neonates is empiric, and there are no guidelines for appropriate cerebral blood pressure management. Some studies have reported an association between hypotension because of neonatal sepsis and PWMI
      • Chau V.
      • Poskitt K.J.
      • McFadden D.E.
      • et al.
      Effect of chorioamnionitis on brain development and injury in premature newborns.
      ; other studies have reported neonatal sepsis alone to be associated with PWMI.
      • Silveira R.C.
      • Procianoy R.S.
      • Dill J.C.
      • da Costa C.S.
      Periventricular leukomalacia in very low birth weight preterm neonates with high risk for neonatal sepsis.
      In our study, we did not find clinical chorioamnionitis, histologic chorioamnionitis/funisitis or positive blood, cerebrospinal or urine cultures to be significantly more common in neonates with PWMI; however, our sample size may not be large enough to detect these possible differences. Although our sample size was large enough to find significant differences in GFAP levels based on head ultrasound scanning abnormalities, we lacked adequate power to determine differences in GFAP based on the later diagnosis of cerebral palsy. Although there was a trend towards those neonates with cerebral palsy having higher GFAP levels, a larger sample size may have allowed us to detect statistically significant differences.
      This study addresses one of the major problems in the care of preterm infants; that many now survive, but with neurological deficits. Although a head ultrasound scan that is performed at 6 weeks of life is the traditional tool used to identify infants with PWMI, a brain-specific biomarker such as GFAP that could identify these infants shortly after birth would allow their identification and triage for therapy much earlier. A rapid perinatal test for neonatal brain injury would have great clinical utility in the identification of infants who could benefit from investigational treatments for PWMI, the quantification of therapeutic efficacy, and the provision of early prognostic information.

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