JOURNAL OF NEUROTRAUMAVolume 20, Number 8, 2003© Mary Ann Liebert, Inc.

Short Communication

F2-Isoprostane and Neuron-Specific Enolase in Cerebrospinal Fluid after Severe Traumatic Brain Injury

in Infants and Children

SUMEETA VARMA,1 KERI L. JANESKO,1 STEPHEN R. WISNIEWSKI,4 HÜLYA BAYIR,1,2

P. DAVID ADELSON,3 NEAL J. THOMAS,5 and PATRICK M. KOCHANEK1,2

ABSTRACT

It has been hypothesized that oxidative stress plays an important role in mediating secondary dam-age after traumatic brain injury (TBI). To study the relationship between lipid peroxidation, clini-cal variables, and neuronal damage in pediatric TBI, we measured levels of F2-isoprostane, a markerof lipid peroxidation, and neuron-specific enolase (NSE), a marker of neuronal damage, in serialcerebrospinal fluid (CSF) samples from 23 infants and children with severe TBI (Glasgow ComaScale score , 8). These were compared to CSF samples from 10 uninjured pediatric controls. Ond1 after injury, F2-isoprostane was increased 6-fold vs. control (36.59 6 8.96 pg/ml vs. 5.64 6 8.08pg/ml, p 5 0.0035) and NSE was increased 10-fold (100.62 6 17.34 ng/ml vs. 8.63 6 2.76 ng/ml, p 50.0002). Multivariate analysis of F2-isoprostane levels and selected clinical variables showed a trendtoward an inverse association with time after injury (p 5 0.0708). Multivariate analysis of NSE lev-els and selected variables showed a positive association between d1 NSE and F2-isoprostane (p 50.0426). CSF F2-isoprostane increases early after TBI in infants and children and is correlated withNSE, supporting a role for oxidative stress in the evolution of secondary damage early after severeTBI in infants and children.

Key words: child abuse; free radical; head injury; inflicted childhood trauma; oxidative stress; pediatric

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1Safar Center for Resuscitation Research, 2Departments of Critical Care Medicine, 3Neurosurgery, 4Epidemiology and PublicHealth, University of Pittsburgh School of Medicine, Pittsburgh, and 5Department of Pediatrics, Division of Pediatric CriticalCare Medicine, Penn State Children’s Hospital and Pennsylvania State University College of Medicine, Hershey, Pennsylvania.

INTRODUCTION

TRAUMATIC BRAIN INJURY (TBI) initiates numerousbiochemical, cellular, and molecular cascades that

are implicated in secondary damage in infants and chil-dren (Kochanek et al., 2000). Based on evidence from

experimental models of central nervous system (CNS) in-jury, production of free radicals and resultant oxidativestress are involved in many of these cascades, includingexcitotoxicity and programmed cell death (Pellegrini-Gi-ampietro et al., 1990; Cheng and Sun, 1994; Dugan etal., 1995; Fujimura et al., 1999), and are likely to play

an important role in mediating secondary damage afterTBI. Free radicals are highly reactive chemical speciesthat in vivo can oxidize lipids, DNA, and proteins, lead-ing to widespread damage. The brain is especially sus-ceptible to oxidative stress because of its high rate ofmetabolic activity, which produces free radical interme-diates, and its low levels of endogenous antioxidants(Ikeda and Long, 1990). The high membrane-to-cyto-plasm ratio and prevalence of polyunsaturated fatty acidsin neuronal membranes make the brain particularly vul-nerable to lipid peroxidation.

When arachidonic acid-containing lipids are oxidizedby free radicals, the products include F2-isoprostanes, afamily of prostaglandin F2-like compounds. These stable,specific products of free radical-catalyzed lipid peroxi-dation are reliable markers of oxidative stress in vivo(Roberts and Morrow, 2000). F2-isoprostanes are initiallyformed in situ on membrane lipids, are released by phos-pholipases and can then be detected in biological fluids,including cerebrospinal fluid (CSF) (Morrow et al.,1992). Increases in F2-isoprostanes have supported freeradical involvement in many CNS diseases (Practico etal., 1998; Montine et al., 1999; Marin et al., 2000), in-cluding TBI (Tyurin et al., 2000).

In a preliminary clinical study by our group in six pe-diatric patients with severe TBI using a comprehensivebattery of markers of oxidative stress and antioxidant re-serve, we noted a transient increase in CSF F2-iso-prostanes early after TBI (Bayi·r et al., 2002). The pre-sent study attempts to confirm this finding in a largersample of pediatric TBI patients, drawn from two cen-ters, and also to extend it by examining any relationshipbetween F2-isoprostane levels and key clinical variables,and the extent of neuronal damage as quantified by neu-ron-specific enolase (NSE), a neuronal marker, in theCSF.

MATERIALS AND METHODS

This study was approved by the IRBs of the Univer-sity of Pittsburgh and Penn State Children’s Hospital andconsent was obtained. Patient samples were coded andpatient names maintained in a locked file to ensure con-fidentiality. A total of 33 children were enrolled in thisstudy. Twenty-three had severe TBI (Glasgow ComaScale [GCS] score , 8 at any time after injury): 17 wereadmitted to the pediatric intensive care unit (PICU) ofChildren’s Hospital of Pittsburgh, and 6 to the PICU ofPenn State Children’s Hospital. Ventricular CSF wasdrained from each patient as part of standard of care. Weassessed 85 CSF samples collected from these patients atthe time of catheter placement and at various timepoints

until catheter removal. Demographic and clinical infor-mation included age, gender, admission GCS score,mechanism of injury (inflicted vs. non-inflicted), and out-come (survival vs. non-survival) at PICU discharge. CSFsamples were also obtained from ten pediatric patientswho underwent lumbar puncture to rule out meningitis.No control patients had evidence of meningitis, TBI, hy-poxia-ischemia, or seizures.

After collection, CSF samples were centrifuged for 10min to remove cellular debris, and then frozen at 270°C until analysis. F2-isoprostane concentrations were mea-sured in CSF samples obtained on d 1, d 2, and d 31

from injured patients, and single samples from uninjuredcontrols. Measurements were made using enzyme im-munoassay kits (Cayman Chemical, Ann Arbor, MI,lower limit of detection of 5 pg/ml). In our initial report(Bayi·r et al., 2002), F2-isoprostanes peaked during theinitial 24 h after TBI. Thus, to assess the relationship be-tween F2-isoprostanes and a biochemical marker of in-jury severity, NSE concentrations were measured in thed 1 CSF samples from injured patients and controls. NSEwas measured using enzyme immunoassay kits (SynXPharma Inc, Ontario, Canada) with a lower limit of de-tection of 1 ng/ml.

Data are expressed as mean 6 SEM. Results were an-alyzed using the Mann-Whitney U-test, Pearson correla-tion, and multivariate regression models. Statistical sig-nificance was set at a p-value , 0.05.

RESULTS

A total of 95 samples were analyzed from 33 patients.In the injury group, age ranged from 0.2 to 15 years. Ten(43%) of the patients were #4 years of age. Control pa-tients ranged in age from 0.8 to 10 years, with six (60%)#4 years of age. In the injury group there were 18 sur-vivors, 4 nonsurvivors, and one patient for whom out-come was not determined. Six of the children studiedwere diagnosed with inflicted TBI (shaken baby syn-drome), while the remaining non-inflicted injuries weredue to a variety of accidental causes (Table 1).

CSF F2-isoprostane was increased between five- andsix-fold vs. control on d1 after injury (36.59 6 8.96 pg/mlvs. 5.6468.08 pg/ml, p 5 0.0035). This increase was be-ginning to resolve by d 2. Although we showed an ,3-fold higher mean F2-isoprostane level on d 2 (vs. con-trol), the elevation did not reach significance (Fig. 1).Multivariate analysis including F2-isoprostane, age, gen-der, admission GCS, mechanism of injury, time, and out-come revealed a trend toward higher levels early after in-jury (p 5 0.0748). No other significant relationship wasrevealed.

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Day 1 NSE levels were increased almost ten-fold vs.control (100.62 6 17.34 ng/ml vs. 8.63 6 2.76 ng/ml,p 5 0.0002). F2-isoprostane level was positively corre-lated with NSE, a biochemical marker of neuronal dam-age within the trauma group (r 5 0.44 p 5 0.0427). Thecorrelation remained significant when control patientswere also included in the analysis. Multivariate analysisof select variables on NSE levels also showed a positive

association between d 1 F2-isoprostane and NSE (p 5

0.0426, Table 2). Finally 7 of the 23 patients had multi-ple trauma (4 orthopedic, 2 abdominal, 1 chest); how-ever, there was no difference in CSF isoprostane levelsin patients with vs without multiple trauma on d 1(39.40 6 22.11 vs 36.54 6 10.26 ng/ml, NS).

DISCUSSION

This study is the first to our knowledge that examinesa marker of oxidative stress after pediatric TBI in rela-tion to either clinical variables or a biochemical markerof injury severity. These findings join a growing body ofevidence for a role for oxidative stress in secondary dam-age after TBI. In experimental TBI, superoxide dismu-tase (SOD) (Muir et al., 1995; De Witt et al., 1997) anda variety of antioxidants (Xiong et al., 1997; Wada et al.,1999) have shown protective effects. Through use of spintraps and ESR spectroscopy, the formation of hydroxylradical (Sen et al., 1994) and ascorbyl radical (Awasthiet al., 1997) have been detected after experimental TBI.Further evidence of the role of oxidative stress in exper-imental TBI came from our group’s study using con-

F2-ISOPROSTANE AND NEURON-SPECIFIC ENOLASE IN CSF

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TABLE 1. DEMOGRAPHIC DATA FOR INFANTS AND CHILDREN WITH SEVERE TRAUMATIC BRAIN INJURY

Patient Age Gender Mechanism GCS Outcome

1 5 F MVA 5 Survived2 5 M MVA 7 Survived3 0.3 M MVA 5 Survived4 11 M Fall from bike 4 Survived5 4 M MVA 3 Died6 7 M Fall 3 Died7 3 M MVA 7 Survived8 15 M Bike v. auto 4 Survived9 8 M Hit w/rock 10 Survived10 8 M MVA 9 Survived11 4 M Ped v. auto 7 Survived12 4 M Ped v. auto 7 Survived13 5 F MVA 3 Died14 2 M Abuse 5 Died15 5 M Abuse 6 Survived16 1.8 F Abuse 7 Survived17 0.2 F Abuse 9 Survived18 4.2 F Bike into 3 Survived

parked car19 14 F MVA 5 Survived20 14 M Bike v. auto 5 Survived21 9 F Car v. train 3 —22 0.2 M Abuse 15 Survived23 3 M Abuse 7 Survived

GCS, Glasgow Coma Scale score; MVA, motor vehicle accident; ped, pedestrian.

FIG. 1. CSF F2-isoprostanes after pediatric TBI and in con-trols. *p 5 0.0035. **One outlying level excluded.

temporary markers and methods to study oxidative stressafter TBI in rats (Tyurin et al., 2000). Also, in experi-mental models, the immature brain has been shown to beparticularly vulnerable to oxidative stress; glutathioneperoxidase and catalase levels are low in immature vsadult rat brain (Fullerton et al., 1998).

Confirmation of the role of oxidative stress in clinicalTBI has been more difficult to obtain. The use of eitherSOD (Muizelaar et al., 1993) or tirilazad mesylate (Mar-shall et al., 1998) in severe TBI in adults failed to im-prove outcome. This was suggested to result, in part, fromlimited brain penetration by these agents (Ikeda et al.,1990; Hall et al., 1996). Evidence that oxidative stresswas attenuated by treatment was not included in eitherreport. Our study was undertaken based on our prior workin pediatric patients, which assessed oxidative stress af-ter TBI in a small number of children (using a variety ofCSF assays) that suggested that CSF analysis is an ex-cellent clinical method to assess the biochemical efficacyof therapies targeting oxidative stress (Bayi·r et al., 2002).

CSF F2-isoprostane levels peak in the first 24 h. Thisagrees with our preliminary pediatric study and our find-ings in adults (Bayi·r et al., 2002). The rapid and transientincrease in F2-isoprostanes may result from the highly re-active nature of free radicals. Cristofori et al. (2001) re-ported early increases in CSF malondialdehyde in adultsafter TBI. Studies with radical scavengers and spin trapshave found that reactive oxygen species are producedwithin minutes of injury in experimental TBI (Lewen etal., 2000). Due to their high reactivity, these radicals re-act quickly with surrounding tissue, leading to a rapid buttransient increase in oxidation products like F2-iso-prostanes. Surprisingly, there was no association betweenF2-isoprostane levels and injury mechanism. Childrenwith shaken baby syndrome generally have a worse prog-nosis and more extreme biochemical derangements thanthose accidentally injured (Whalen et al., 1998; Bell et

al., 1999; Ruppel et al., 2001). However, abuse victimsare also more likely to have a delay in presentation (Chad-wick, 1992) that may account for our inability to detecta difference in their F2-isoprostane levels: given the tran-sient nature of the increase we found, we may havemissed the true F2-isoprostane peaks of abuse victimswith delayed presentation. Alternatively, the unique andvariable nature of the insult in child abuse may yield lesspredictable F2-isoprostane levels and time course (Bruceand Zimmerman, 1989). The second highest F2-iso-prostane level we measured was in an abuse victim witha 2–3-d delay in presentation. This suggests that eitherthis patient’s actual peak level was even higher, or thatthe F2-isoprostane level peaked much later than expected.Berger et al. (2002) also reported that the peak in CSFNSE in children with inflicted TBI was delayed in com-parison to children with non-inflicted TBI.

We recently reported markedly lower levels of F2-iso-prostanes in adult women than in men after severe TBI(Bayi·r et al., 2001). In contrast, we saw no gender effecton F2-isoprostane levels in this pediatric study. This wasexpected since the proposed explanation of the gender ef-fect in adults relies on sex hormones. Adult women areprotected from oxidative stress (Proteggente et al., 2002)and antioxidant effects of female sex hormones are seenin experimental CNS injury (Roof and Hall, 2000; Behl,2002).

CSF NSE may be a useful quantitative measure ofbrain injury severity (Uzan et al., 1995; Ross et al., 1996;Berger et al., 2002). Such a marker is useful due to lim-itations of the GCS score to evaluate injury severity inyoung children (Durham et al., 2000). We noted an ,10-fold increase in CSF NSE on d 1. We also found that F2-isoprostane and NSE levels were correlated, supportinga relationship between lipid peroxidation and the extentof neuronal damage. Whether this represents a link be-tween lipid peroxidation and neuronal damage leading to

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TABLE 2. ANALYSIS OF SELECT VARIABLES ON CEREBROSPINAL FLUID NEURON-SPECIFIC ENOLASE LEVELS

Bivariate model Multivariate model

Measure b p b p

Isoprostane 0.8085 0.0427 0.7954 0.0426Age* 249.0850 0.1942 228.3662 0.5538Gender1 260.0631 0.1256 258.4514 0.1287GCS** 17.6897 0.6461 6.6831 0.8686Mechanism2 58.0547 0.1396 26.7334 0.5876

*Age #4 vs. .4.**GCS 3, 4 vs. .4.1The reference group for gender was males.2The reference group for mechanism was non-inflicted.GCS, Glasgow Coma Scale score.

NSE release or a common response of these two mark-ers to the primary injury cannot be determined from thisstudy. Since Berger et al. (2002) showed that abuse vic-tims have a delayed increase in CSF NSE, it may havebeen informative to compare peak levels of F2-iso-prostane and NSE, rather than d 1 levels.

We conclude that there is a rapid and transient increasein F2-isoprostanes, a marker of lipid peroxidation, in theCSF after severe TBI in infants and children. On d 1 af-ter injury, F2-isoprostane levels are correlated with lev-els of NSE, a marker of neuronal damage. These find-ings support a role for oxidative stress in secondaryneuronal damage after TBI in infants and children.

ACKNOWLEDGMENTS

We thank the University of Pittsburgh Center for InjuryResearch and Control (CIRCL)/CDC. Dr. Bayi·r is sup-ported by the Charles Schertz fellowship grant for theDepartment of Anesthesiology and Critical Care Medi-cine at the University of Pittsburgh. Dr. Thomas is sup-ported by the Children’s Miracle Network ResearchGrant, Penn State Children’s Hospital.

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Address reprint requests to:Patrick M. Kochanek, M.D.

Safar Center for Resuscitation ResearchDepartment of Critical Care Medicine

3434 Fifth AvenuePittsburgh, PA 15260

E-mail: kochanekpm@ccm.upmc.edu

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