• Share
  • Email
  • Embed
  • Like
  • Save
  • Private Content


Flash Player 9 (or above) is needed to view presentations.
We have detected that you do not have it on your computer. To install it, go here.

Like this document? Why not share!

Education Research: Cognitive performance is preserved in sleep ...






Total Views
Views on SlideShare
Embed Views



0 Embeds 0

No embeds



Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
Post Comment
Edit your comment

    Education Research: Cognitive performance is preserved in sleep ... Education Research: Cognitive performance is preserved in sleep ... Document Transcript

    • RESIDENT& FELLOWSECTION Education Research:Section Editor Cognitive performance is preserved inMitchell S.V. Elkind,MD, MS sleep-deprived neurology residentsM. Reimann, PhD ABSTRACTR. Manz, MD Objective: To test the hypotheses that sleep deprivation in neurology residents is associated withS. Prieur performance deficits and that vigilance and cognitive performance is more compromised afterH. Reichmann, Prof overnight on-call duty compared to night shift.T. Ziemssen, MD Methods: Thirty-eight neurology residents of a university teaching hospital participated in a prospec- tive single-blind comparison study. Residents were recruited according to their working schedule andAddress correspondence and divided into 3 groups: 24 hours overnight on-call duty, night shift, and regular day shift (controls). Allreprint requests to Dr. Manja participants underwent serial measurements of sleepiness and cognitive performance in the morningReimann, ANF Laboratory, directly after or before their shift. Pupillary sleepiness test and Paced Auditory Serial Addition TestDepartment of Neurology,University Clinic Carl Gustav were applied. Perceived sleepiness was assessed by a questionnaire.Carus, Dresden University ofTechnology, Fetscherstraße 74, Results: Sleepiness was increased in residents after night shift and overnight call compared toD-01307 Dresden, Germany controls while the type of night duty was not associated with the extent of sleepiness. Sleep-manjareimann@uniklinikum-dresden.de deprived residents did not show any performance deficits on the Paced Auditory Serial Addition Test. Cognitive performance was not associated with sleepiness measures. Conclusions: Night shift and overnight call duty have a similar impact on alertness in neurology residents. Sleep-deprived neurology residents may be able to overcome sleep loss–related per- formance difficulties for short periods. Neurology® 2009;73:e99 –e103 GLOSSARY PASAT Paced Auditory Serial Addition Test; PST Pupillography Sleepiness Test; PUI pupillary unrest index; SSS self-stated sleepiness. Despite recent changes in working schedule regulation for clinicians, long working hours remain a common feature in health care.1-3 Associated sleep deprivation and fatigue present not only a serious concern for patient safety, but also places the health of health care professionals at risk. Serious problems resulting from sleep loss range from performance deficits and erroneous decision making to increased risk for motor vehicle accidents.2,4 The majority of sleepiness studies have been performed in certain medical specialties with a reputation for demanding schedules, such as surgery or intensive care.2 Neurologists are frequently underrated in terms of intensity and heaviness of their working schedules due to their mistakenly close relationship to psychiatry. Call rotation on the neurology ward and night shifts at the neurol- ogy intensive care unit are as common as in internal medicine or surgery. Actually, neurologists are frequently challenged by the high prevalence of life-threatening strokes in the Western society, where any delay in action or poor performance may be fatal for the patient. This prompted us to investigate sleepiness and cognitive performance in sleep-deprived and alert neurologists working at a large university neurology clinic. We hypothesized that 1) sleep depriva- tion in neurologists is associated with performance deficits as previously demonstrated for other From the Autonomic and Neuroendocrinological Laboratory, Department of Neurology (M.R., R.M., S.P., H.R., T.Z.), and Research Group Neuro- Metabolism, Department of Neurology and Internal Medicine III (M.R., T.Z.), University Hospital Carl Gustav Carus, Dresden University of Technology, Germany. Supported by the University Hospital Carl Gustav Carus, Dresden, Germany. Disclosure: Author disclosures are provided at the end of the article. Copyright © 2009 by AAN Enterprises, Inc. e99
    • medical specialties and that 2) vigilance as well due to sleep on a 5-point Likert scale. All measurements were serially repeated up to a maximum of 13 times. Measurement frequency, as cognitive performance is more compromised however, varied between individuals according to the rotation after 24 hours overnight on-call duty compared schedules (4.8 3.3). to night shift. Pupillary Sleepiness Test. We performed the PST (AMTech, Dossenheim, Germany) in a quiet and darkened room after an ini- METHODS This pilot study included 38 neurology residents tial dark-adapting phase of 15 minutes. During PST, residents wore (19 women and 19 men, mean age 30 2 years) recruited from the goggles equipped with infrared light transmitting filter glasses im- Department of Neurology at the University Hospital Carl Gustav pervious to visible light. They were seated on a comfortable chair Carus, Dresden, Germany. We screened residents for exclusion cri- and head position was adjusted by a chin rest fixed on a table. An teria such as current use of medication known to affect the sleep/ infrared video camera was fixed at a distance of 70 cm from the wake cycle or daytime alertness, current psychiatric illness, and sleep examination subject. We instructed the clinicians to maintain fixa- disorder diagnosis. We then stratified eligible residents according to tion on a set of infrared light-emitting diodes. We then recorded their working schedule into 3 groups: group 1, 24 hours overnight spontaneous pupillary oscillations over a period of 11 minutes by on-call duty; group 2, night shift; and group 0, regular day shift infrared video pupillography and evaluated the recording by 25-Hz (control). real-time analysis as published elsewhere.5 Pupillary unrest index Definitions of groups. Residents in group 1 (24 hours over- (PUI) is a measure of pupillomotor hippus in darkness and calcu- night on-call duty) performed their regular shift from 8 AM– 4 lated as an integrated sum of slow movements of the pupillary mar- PM followed by overnight call until 8 AM the next day. During gin during the measurement period.6 This value is usually low in their overnight duty, they were allowed to sleep but there was no alertness and increases with progressive sleepiness. We also calcu- scheduled coverage. Residents have usually 3 to 4 overnight calls lated the mean pupil diameter over the entire recording period of 11 per month. Residents in group 2 (night shift) worked at the minutes. During sleepiness the initial diameter is reduced and the intensive care unit for 7 consecutive days daily from 8 PM to 8 AM mean pupil size falls below the initial diameter toward the end of the the following morning. Afterwards they had 1 week off. Sleeping measurement. was not permitted during their shifts. From experience, residents Paced Auditory Serial Addition Test. The validated and have on average 2 admissions and 30 internal and 3 outpatient computer-aided PASAT allows for measuring the capacity and consultations per night. Night shift rotation was every 5 to 6 velocity of information processing within the auditory-verbal do- weeks over a period of 1 year. Residents in group 0 (day shift) main (cognitive performance).7,8 The test system entails the sub- regularly worked from 7:30 AM to 3:30 PM. However, residents ject to continuously add the last 2 numbers of consecutive series on day shift frequently work overtime. and to announce the sum aloud. Numbers from 1 to 9 are an- Standard protocol approvals, registrations, and patient con- nounced acoustically in random order by a PC with the screen sents. The Ethics Committee on human experimentation of the remaining dark. To avoid practice effects, clinicians were trained Dresden University of Technology approved the study and the in- on the PASAT at least 3 times before commencing the study. We vestigation conformed with the principles outlined in the Declara- applied the 60-item short version of the test (maximal score of tion of Helsinki. We informed all participating residents about the 60). Lower scores (small numbers of correct answers) indicate objectives and procedures of the study and obtained written in- worse cognitive performance. formed consent prior to their inclusion. The serial data collection Statistical analysis. We used the SPSS software package ver- was performed at the Autonomic and Neuroendocrinological Labo- sion 16.0 for Windows (SPSS Inc., Chicago, IL) for all statistical ratory of the University Clinic. We measured all residents before 9 evaluation. Data are presented as median and 25th–75th percen- AM directly after their (night) shift rotation or just before day shift tile unless otherwise stated. Owing to the small sample size we commenced (controls). The assistant performing the measurements assumed non-Gaussian distribution, and hence applied nonpara- was blinded. We measured objective sleepiness by Pupillography metric tests with Bonferroni correction for comparing groups. Sleepiness Test (PST) and cognitive performance by Paced Audi- Spearman correlation coefficients were calculated. A two-tailed tory Serial Addition Test (PASAT). We instructed the residents to p 0.05 was regarded as the level of significance. abstain from drinking alcoholic beverages, smoking, and drinking coffee for at least 4 hours before the measurements. The residents rated their sleepiness on a 5-point Likert scale based on the state- RESULTS Before we started the comparison among ment “Currently I feel.” We also recorded the number of hours slept the 3 groups, we assessed the strength of association in the previous 24 hours and assessed the perceived recovery effect between the first measurement and the mean of serial Table Sleepiness and cognitive performance of neurology residents by type of night duty 2 Parameters Overnight call (n 17) Night shift (n 6) Control (n 15) (p*) Pupil diameter (mm) 7.51 (6.25–7.75) 7.23 (5.18–7.54) 7.21 (6.61–8.00) 0.59 (0.744) Pupillary unrest index (mm/min) 7.00 (4.96–9.44)a 10.34 (7.78–15.07)a 4.72 (3.86–5.09)b 12.88 (0.002) PASAT score† 56 (49–58) 51 (42–58) 54 (48–56) 0.67 (0.715) Self-stated sleepiness‡ 2.3 (1.7–2.7)a 2.6 (2.2–3.1)a 1.0 (0–1.0)b 20.24 ( 0.001) Data are median (interquartile range). Unequal superscript letters indicate significant differences (Mann-Whitney U test). *Kruskal-Wallis test. †Paced Auditory Serial Addition Test (number of correct answers/60). ‡Categorical value.e100 Neurology 73 November 24, 2009
    • average a mean (minimum–maximum) of 2 (1–3)Figure 1 Self-stated sleepiness of neurology residents by type of night duty admissions, 2 (0 –7) consultations, and 3 (1–5) tele- phone inquiries during overnight on-call duty. Figure 1 illustrates the proportion of respective responses to the statement “Currently I feel . . .” Fig- ure 2 depicts the self-stated recovery effect due to sleep in the 24 hours preceding the examination. Correlation analyses did not reveal any association between the PUI and the PASAT score. However, the PUI increased (r 0.507, p 0.001) and the PASAT score decreased (r 0.335, p 0.04) with increased SSS in the total sample. The perceived level of sleepiness decreased as the number of sleeping hours in the past 24 hours increased (r 0.527, p 0.001). The above associations could not be confirmed in subgroups (p 0.05). measurements. We revealed significant correlations DISCUSSION Rotating shift work in clinics to pro- with r values ranging from 0.763 to 0.904 ( p vide 24-hour patient care has come increasingly under 0.001). Based on these results, we continued our scrutiny due to negative effects associated with sleep analysis using the mean values. loss, fatigue, and circadian disruption.9,10 Although PUI and self-stated sleepiness (SSS) were signifi- night shift and 24 hours overnight on-call duty consid- cantly affected by type of night duty while pupil diame- erably differ in terms of number of working hours, per- ter and the PASAT score remained unaffected (table). It mission for midshift naps, and rotation frequency, their appeared that PUI and SSS were significantly higher effect on sleepiness and cognitive performance has never after the night shift and the 24 hours overnight on-call been distinguished. We hypothesized that residents on duty compared to a normal night at home. Residents 24 hours call rotation would be more affected by sleep after night shift and 24 hours overnight on-call duty did loss due to a longer and more irregular working sched- not differ with respect to sleepiness measures. ule. We investigated this hypothesis in neurology resi- Neurology residents on 24 hours overnight on- dents of a large university clinic. This specialty group is call duty had slept on average 4.3 (2.8 – 4.6) hours often underrated in terms of heaviness and intensity of (midshift nap) in the last 24 hours, which was signif- labor and therefore has never been investigated in sleep- icantly less compared to their colleagues on night iness studies. Importantly, previous sleepiness studies in shift (5.9 [4.9 –7.0] hours, p 0.006) or on day shift selected medical specialties explicitly emphasized that (controls) (6.5 [6.0 –7.0] hours, p 0.001). The results must not be extrapolated to other medical spe- longest sleeping phase during 24 hours overnight on- cialty groups.3,4,11 Although we could not verify the call duty was 3.0 (2.0 –3.8) hours. Residents had on above hypothesis, our results clearly demonstrate that sleepiness is a common problem among neurology resi-Figure 2 Self-stated recovery effect due to sleep in the past 24 hours dents undergoing night shift and 24 hours overnight on-call duty. We additionally demonstrated that vigilance mea- sured by PST is in good agreement with SSS. This finding corresponds with previous studies suggesting the PST as a valid and objective tool to detect sleepi- ness in healthy subjects.6 The lack of significant performance decrements in sleep-deprived neurology residents vs controls is intrigu- ing since an association between performance and acute sleep deprivation was found in previous studies.2,4,12 Differences in study design, medical specialty, and methods for vigilance testing limit comparisons across studies. However, one study also failed to show a signif- icant performance decrement on the complex PASAT test in sleep-deprived normal subjects.13 The investiga- tors concluded that university-based research may func- Neurology 73 November 24, 2009 e101
    • tion as a motivational incentive, which tends to offset ACKNOWLEDGMENT sleepiness effects on performance.13 The authors thank all neurology residents for their participation in this study. Alternatively, the applied method for performance testing may have been suboptimal for our population DISCLOSURE since its clinical utility is mainly proven in neuropsycho- Dr. Reimann, Dr. Manz, and S. Prieur report no disclosures. Dr. Reich- mann serves on scientific advisory boards, receives speaker honoraria, logical syndromes. However, more recent studies also and/or receives funding for travel from Cephalon, Inc., Novartis, Teva employed the PASAT in healthy adults with satisfactory Pharmaceutical Industries Ltd., Lundbeck Inc., GlaxoSmithKline, Boehr- results.7,14 Originally assumed to measure rate of infor- inger Ingelheim, Bayer Schering Pharma., UCB/Schwarz Pharma, Desitin Pharmaceuticals, GmbH, Pfizer Inc., and Solvay Pharmaceuticals, Inc. mation processing, the PASAT is now recognized as Dr. Ziemssen has received speaker honoraria from Biogen Idec, Sanofi- tapping into different types of cognitive processes.15 Aventis, Merck Serono, Novartis, Teva Pharmaceutical Industries Ltd., This multifactorial nature may complicate the interpre- and Bayer Schering Pharma; serves as a consultant for Teva Pharmaceuti- cal Industries Ltd., Novartis, and Bayer Schering Pharma; and receives tation of test result from sleep-deprived subjects espe- research support from the Roland Ernst Foundation. cially under the assumption that sleep loss affects different cognitive pathways differentially. The consis- REFERENCES tently high PASAT score across all study groups sug- 1. Reddy R, Guntupalli K, Alapat P, Surani S, Casturi L, gests that sleep-deprived neurology residents are able to Subramanian S. Sleepiness in medical ICU residents. overcome performance difficulties for a limited time pe- Chest 2009;135:81– 85. riod. This assumption seems plausible since stressful 2. Veasey S, Rosen R, Barzansky B, Rosen I, Owens J. Sleep working situations are routine in the clinic and thus loss and fatigue in residency training: a reappraisal. JAMA may facilitate partial conditioning. Although there is ev- 2002;288:1116 –1124. 3. Surani S, Subramanian S, Aguillar R, Ahmed M, Varon J. idence that sleep-deprived resident doctors are prone to Sleepiness in medical residents: impact of mandated reduc- committing medical errors,4,9 tasks of short duration tion in work hours. Sleep Med 2007;8:90 –93. may be less likely to detect performance deficits in 4. Arnedt JT, Owens J, Crouch M, Stahl J, Carskadon MA. chronically sleep deprived individuals.2 Neurobehavioral performance of residents after heavy night Importantly, we adequately controlled for prac- call vs after alcohol ingestion. JAMA 2005;294:1025–1033. 5. Ludtke H, Wilhelm B, Adler M, Schaeffel F, Wilhelm H. tice effects8 first by using a control group and sec- Mathematical procedures in data recording and processing of ondly by training the clinicians on the PASAT prior pupillary fatigue waves. Vision Res 1998;38:2889 –2896. to the study. Consequently, we obtained a very good 6. Wilhelm B, Wilhelm H, Ludtke H, Streicher P, Adler M. agreement between the first measurement and the Pupillographic assessment of sleepiness in sleep-deprived mean of serial measurements. In addition, major healthy subjects. Sleep 1998;21:258 –265. confounders of the PASAT such as age, gender, edu- 7. Diehr MC, Cherner M, Wolfson TJ, Miller SW, Grant I, Heaton RK. The 50 and 100-item short forms of the cation, and ethnicity14,16 were accounted for by creat- Paced Auditory Serial Addition Task (PASAT): demo- ing a homogenous study sample. graphically corrected norms and comparisons with the full Nevertheless, it must be considered that our results PASAT in normal and clinical samples. J Clin Exp Neuro- are compromised by a questionable rested control psychol 2003;25:571–585. group. Correspondingly, the average amount of sleep in 8. Tombaugh TN. A comprehensive review of the Paced Au- ditory Serial Addition Test (PASAT). Arch Clin Neuro- our controls was equivalent to the accepted core sleep psychol 2006;21:53–76. requirement of 6.5 hours (many were also below).17 In 9. Barger LK, Cade BE, Ayas NT, et al. Extended work shifts addition, nearly two thirds of the controls felt fairly to and the risk of motor vehicle crashes among interns. poorly rested, which indicates chronic partial sleep de- N Engl J Med 2005;352:125–134. privation (figure 3). This finding is relevant as chronic 10. Gold DR, Rogacz S, Bock N, et al. Rotating shift work, partial sleep deprivation appears to be cumulative with sleep, and accidents related to sleepiness in hospital nurses. Am J Public Health 1992;82:1011–1014. respect to performance decrements.18 11. Saxena AD, George CF. Sleep and motor performance in on- Neurology residents on night shift and overnight call internal medicine residents. Sleep 2005;28:1386 –1391. call are affected to a similar extent by sleepiness. In- 12. Grantcharov TP, Bardram L, Funch-Jensen P, Rosenberg creased sleepiness, however, did not affect performance J. Laparoscopic performance after one night on call in a on the complex PASAT test. It seems that sleep- surgical department: prospective study. BMJ 2001;323: 1222–1223. deprived neurology residents may be able to overcome 13. Hood B, Bruck D. A comparison of sleep deprivation and sleep loss–related performance difficulties for short peri- narcolepsy in terms of complex cognitive performance and ods. This, however, may not necessarily apply for more subjective sleepiness. Sleep Med 2002;3:259 –266. demanding procedures outside the laboratory. 14. Diehr MC, Heaton RK, Miller W, Grant I. The Paced Auditory Serial Addition Task (PASAT): norms for age, education, and ethnicity. Assessment 1998;5:375–387. AUTHOR CONTRIBUTIONS 15. Madigan NK, DeLuca J, Diamond BJ, Tramontano G, Statistical analysis was conducted by Dr. Manja Reimann. Averill A. Speed of information processing in traumatice102 Neurology 73 November 24, 2009
    • brain injury: modality-specific factors. J Head Trauma Re- 17. Bonnet MH, Arand DL. We are chronically sleep de- habil 2000;15:943–956. prived. Sleep 1995;18:908 –911.16. Wills S, Leathem J. The effects of test anxiety, age, intelli- 18. Dinges DF, Pack F, Williams K, et al. Cumulative sleepi- gence level, and arithmetic ability on Paced Auditory Serial ness, mood disturbance, and psychomotor vigilance per- Addition Test performance. Appl Neuropsychol 2004;11: formance decrements during a week of sleep restricted to 180 –187. 4 –5 hours per night. Sleep 1997;20:267–277. Neurology 73 November 24, 2009 e103
    • The n e w e ng l a n d j o u r na l of m e dic i n e original article Moderate Hypothermia to Treat Perinatal Asphyxial Encephalopathy Denis V. Azzopardi, F.R.C.P.C.H., Brenda Strohm, R.G.N., A. David Edwards, F.Med.Sci., Leigh Dyet, M.B., B.S., Ph.D., Henry L. Halliday, F.R.C.P.H., Edmund Juszczak, M.Sc., Olga Kapellou, M.D., Malcolm Levene, F.Med.Sci., Neil Marlow, F.Med.Sci., Emma Porter, M.R.C.P.C.H., Marianne Thoresen, M.D., Ph.D., Andrew Whitelaw, F.R.C.P.C.H., and Peter Brocklehurst, F.F.P.H., for the TOBY Study Group* A bs t r ac tBackgroundWhether hypothermic therapy improves neurodevelopmental outcomes in newborn From the Division of Clinical Sciencesinfants with asphyxial encephalopathy is uncertain. and Medical Research Council (MRC) Clinical Sciences Centre, Hammersmith Hospital, Imperial College London, Lon-Methods don (D.V.A., A.D.E., L.D., O.K., E.P.); theWe performed a randomized trial of infants who were less than 6 hours of age and National Perinatal Epidemiology Unit, Uni- versity of Oxford, Oxford (B.S., E.J., P.B.);had a gestational age of at least 36 weeks and perinatal asphyxial encephalopathy. the Department of Perinatal Medicine,We compared intensive care plus cooling of the body to 33.5°C for 72 hours and in- Royal Maternity Hospital and Depart-tensive care alone. The primary outcome was death or severe disability at 18 months ment of Child Health, Queen’s University, Belfast (H.L.H.); the University of Leedsof age. Prespecified secondary outcomes included 12 neurologic outcomes and 14 and Leeds General Infirmary, Leeds (M.L.);other adverse outcomes. the Academic Division of Child Health, Queen’s Medical Centre, Nottingham (N.M.); and the Department of ClinicalResults Science, University of Bristol, St. Michael’sOf 325 infants enrolled, 163 underwent intensive care with cooling, and 162 under- Hospital (M.T.) and Southmead Hospitalwent intensive care alone. In the cooled group, 42 infants died and 32 survived but (A.W.), Bristol — all in the United King- dom. Address reprint requests to Dr. Az-had severe neurodevelopmental disability, whereas in the noncooled group, 44 in- zopardi at the Division of Clinical Scienc-fants died and 42 had severe disability (relative risk for either outcome, 0.86; 95% es and MRC Clinical Sciences Centre,confidence interval [CI], 0.68 to 1.07; P = 0.17). Infants in the cooled group had an Imperial College London, Hammersmith Hospital, Du Cane Rd., London W12 0NN,increased rate of survival without neurologic abnormality (relative risk, 1.57; 95% CI, United Kingdom, or at d.azzopardi@1.16 to 2.12; P = 0.003). Among survivors, cooling resulted in reduced risks of cere- imperial.ac.uk.bral palsy (relative risk, 0.67; 95% CI, 0.47 to 0.96; P = 0.03) and improved scores on *The members of the Total Body Hypo-the Mental Developmental Index and Psychomotor Developmental Index of the Bayley thermia for Neonatal Encephalopathy TrialScales of Infant Development II (P = 0.03 for each) and the Gross Motor Function (TOBY) study group are listed in the Ap-Classification System (P = 0.01). Improvements in other neurologic outcomes in the pendix.cooled group were not significant. Adverse events were mostly minor and not associ- N Engl J Med 2009;361:1349-58.ated with cooling. Copyright © 2009 Massachusetts Medical Society.ConclusionsInduction of moderate hypothermia for 72 hours in infants who had perinatal as-phyxia did not significantly reduce the combined rate of death or severe disabilitybut resulted in improved neurologic outcomes in survivors. (Current Controlled Trialsnumber, ISRCTN89547571.) n engl j med 361;14 nejm.org october 1, 2009 1349 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • The n e w e ng l a n d j o u r na l of m e dic i n e P erinatal asphyxial encephalopathy birth, acidosis (defined as any occurrence of um- is associated with high morbidity and mor- bilical-cord, arterial, or capillary pH of <7.00 or tality rates worldwide and is a major burden base deficit of ≥16 mmol per liter). In addition, for the patient, the family, and society. There is an they had to have moderate-to-severe encephalopa- urgent need to improve outcomes in affected in- thy (indicated by lethargy, stupor, or coma) and fants. either hypotonia, abnormal reflexes (including oc- Experimentally, reducing body temperature to ulomotor or pupillary abnormalities), an absent or 3 to 5°C below the normal level reduces cerebral weak suck, or clinical seizures. Finally, they had injury and improves neurologic function after as- to have abnormal background activity of at least phyxia.1-6 Preliminary clinical studies have found 30 minutes’ duration or seizures on amplitude- no serious adverse effects of cooling.7-9 Two ran- integrated electroencephalography.18 domized, controlled trials, the CoolCap trial10 and We excluded infants expected to be more than the National Institute of Child Health and Hu- 6 hours of age at the time of randomization and man Development (NICHD) trial,11 have reported those with major congenital abnormalities known outcomes among infants at 18 months of age who at randomization that required surgery or were had asphyxial encephalopathy, after slightly dif- suggestive of chromosomal anomaly or syndromes ferent cooling regimens. Only the NICHD trial that involve brain dysgenesis. showed a significant reduction in the composite Written informed consent was obtained from primary outcome of death or disability with hypo- a parent of each infant after explanation of the thermia. Neither trial had sufficient power to de- study, and consent was reaffirmed within the sub- tect significant differences in important individu- sequent 24 hours.19 Assignment to a treatment al neurologic outcomes, and several systematic group was performed by means of central tele- reviews and an expert workshop did not reach a phone randomization or a secure Web-based sys- consensus in recommending hypothermia as stan- tem (provided by the National Perinatal Epide- dard treatment.12-17 miology Unit Clinical Trials Unit, Oxford, United To clarify the role of hypothermia, we carried Kingdom). Minimization was used to ensure bal- out the Total Body Hypothermia for Neonatal En- ance of treatment assignment among infants with cephalopathy Trial (TOBY), a multicenter, random- various grades of abnormality on amplitude-inte- ized trial comparing intensive care plus total-body grated electroencephalography and within each cooling for 72 hours with intensive care without participating center. cooling among term infants with asphyxial en- cephalopathy. Clinical Management All recruited infants were cared for in partici- Me thods pating centers. Infants from referring hospitals were assessed by trained retrieval teams who per- The TOBY protocol was approved by the London formed amplitude-integrated electroencephalog- Multicenter Research Ethics Committee and the raphy, sought consent if the infant was eligible, local research ethics committee of each participat- performed randomization, and for infants assigned ing hospital. Conduct of the study was overseen to the cooled group, began cooling by discontinu- by an independent trial steering committee with ing warming and applying cooled gel packs, if nec- advice from an independent data monitoring and essary, until the infant was admitted to a partici- ethics committee. pating center. To minimize potential confounding from dif- Study Design and Procedures ferential use of cointerventions, uniform guidance TOBY was a randomized, controlled trial, involv- was provided on ventilatory and circulatory care, ing term infants, comparing intensive care plus management of seizures, sedation, and fluid re- total-body cooling for 72 hours with intensive care quirements. All infants underwent sedation with without cooling. Infants were eligible if they were morphine infusions or with chloral hydrate if they born at or after 36 completed weeks’ gestation. appeared to be distressed. Skin temperature and They also had to have, at 10 minutes after birth, rectal temperature (measured at least 2 cm within either an Apgar score of 5 or less or a continued the rectum) were monitored continuously and re- need for resuscitation or, within 60 minutes after corded hourly in all infants throughout the inter-1350 n engl j med 361;14 nejm.org october 1, 2009 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • Moder ate Hypothermia for Perinatal Asphyxial Encephalopathyvention period. Clinical staff were made aware of topenia, major venous thrombosis, renal failurethe treatment assignments so that they could man- treated with dialysis, pneumonia, pulmonary airage cooling appropriately. leak, and duration of hospitalization. (Most out- comes are defined in the Supplementary Appen-Intensive Care Alone dix, available with the full text of this article atInfants assigned to the noncooled group received NEJM.org.)the current standard of care and were placed un- Secondary outcomes at 18 months, specifiedder radiant heaters or in incubators, which were before data analysis, included death and severeservo-controlled according to the abdominal skin neurodevelopmental disability (components of thetemperature to maintain the rectal temperature composite primary outcome), the score on theat 37.0±0.2°C. Psychomotor Developmental Index of BSID-II (on which the standardization mean [±SD] is 100±15Intensive Care with Cooling and higher scores indicate better performance),Infants assigned to the cooled group were treated cerebral palsy, hearing loss, seizures treated within incubators with the power turned off. Hypo- anticonvulsant agents, microcephaly (i.e., age- andthermia was maintained by nursing the infant on sex-standardized head circumference of more thana cooling blanket in which fluid whose tempera- 2 SD below the mean), multiple neurodevelop-ture was regulated by a manually adjusted thermo- mental abnormalities (i.e., more than one of thestat (Tecotherm TS 200, Tec-Com) was circulated. following: a GMFCS score of 3 to 5, a score of <70The target rectal temperature was 33 to 34°C, and on the Mental Developmental Index of BSID-II) (ontypically, the thermostat was set from 25 to 30°C. which the standardization mean [±SD] is 100±15 and higher scores indicate better performance,Rewarming Procedures seizures, or cortical visual impairment and hear-When the period of cooling concluded, 72 hours ing loss), and survival without neurologic abnor-after randomization, the rectal temperature was mality (i.e., a Mental Developmental Index scoremonitored for at least 4 hours to prevent rebound >84, a Psychomotor Developmental Index scorehyperthermia. The rectal temperature was allowed >84, no abnormalities on GMFCS assessment, andto rise by no more than 0.5°C per hour, to a max- normal vision and hearing).imum of 37±0.2°C. Cranial ultrasonography wasperformed daily for the first 4 days after birth, Neurologic Assessmentand magnetic resonance imaging (MRI) was con- Infants were assessed at approximately 18 monthsducted, according to a specified protocol, within 5 of age, through a structured examination by one ofto 14 days after birth. five trained assessors who were unaware of the treatment assignments. Neurologic signs and func-Outcomes tion were scored,20,21 and the presence and typeThe primary outcome at 18 months of age was a of cerebral palsy were determined. Neurodevel-composite of death or severe neurodevelopmental opmental outcome was assessed according to thedisability in survivors. Severe neurodevelopmental BSID-II.22disability was defined as a score of less than 70on the Mental Developmental Index of the Bayley Statistical AnalysisScales of Infant Development II (BSID-II) (on which We estimated that a sample of 236 infants wouldthe standardization mean [±SD] is 100±15 and be required to detect a relative risk of 0.6 to 0.7 forhigher scores indicate better performance), a score the primary outcome in the cooled group as com-of 3 to 5 on the Gross Motor Function Classifica- pared with the noncooled group, with a statisticaltion System20 (GMFCS) (on which scores can range power of 80%, at a two-sided significance level offrom 1 to 5, with higher scores indicating greater 5% and assuming a 10% loss to follow-up. Thisimpairment), or bilateral cortical visual impairment sample size was achieved ahead of schedule, andwith no useful vision. enrollment was continued after the CoolCap and Adverse outcomes included intracranial hemor- NICHD trial results suggested that a larger sam-rhage, persistent hypotension, pulmonary hemor- ple would be valuable.rhage, pulmonary hypertension, prolonged blood Demographic and clinical characteristics werecoagulation time, culture-proven sepsis, necrotiz- summarized at baseline as counts and percentagesing enterocolitis, cardiac arrhythmia, thrombocy- of the total numbers of infants for categorical vari- n engl j med 361;14 nejm.org october 1, 2009 1351 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • The n e w e ng l a n d j o u r na l of m e dic i n e with stratification on the basis of the grade of 494 Patients were assessed for eligibility abnormality on amplitude-integrated electroen- cephalography at randomization and duration of the interval between birth and randomization (0 to 169 Were excluded <4 hours vs. 4 to 6 hours). The consistency of the 94 Did not meet inclusion criteria effect of the treatment group across subgroups 30 Declined to participate was explored by means of the statistical test of 45 Had other reasons interaction. 325 Underwent randomization R e sult s From December 1, 2002, through November 30, 2006, 494 infants were screened and 325 were recruited from 42 hospitals (Fig. 1). The infants 163 Were assigned to undergo 162 Were assigned to undergo were from the United Kingdom (277), Hungary intensive care plus cooling intensive care without cooling (24), Sweden (18), Israel (4), and Finland (2). Base- line characteristics of the infants (including ma- ternal characteristics) were broadly similar between 1 Was lost to follow-up (unable 1 Was lost to follow-up (unable the two groups (Table 1). to collect outcome data) to collect outcome data) 42 Died 44 Died 120 Survivors were assessed 117 Survivors were assessed Compliance with Cooling Protocol Rectal temperatures were similar between the two groups at the time of randomization (Table 1). Mean rectal temperatures at 6 to 72 hours after 163 Were analyzed 162 Were analyzed randomization were 33.5±0.5°C and 36.9±0.6°C in the cooled and noncooled groups, respectively Figure 1. Enrollment and Follow-up of the Study Infants. (Fig. 2). Among the 162 infants who were not AUTHOR: Azzopardi RETAKE 1st cooled, during the treatment period the tempera- ICM REG F ables, as1means (±SD) for normally2nd FIGURE: of 2 distributed ture rose above 38°C on one occasion in 14 (9%) 3rd CASE continuous variables, and as medians and ranges Revised and on more than one occasion in 23 (14%). EMail or interquartile ranges for other SIZE Line 4-C continuous vari- ARTIST: ts H/T H/T Enon ables. 22p3 Primary Outcome Combo DataAUTHOR, PLEASE NOTE: groups to which pa- In the cooled group, 42 infants died and 32 sur- were analyzed in the Figure has been been assigned regardless of either de- vived with severe neurodevelopmental disability, tients had redrawn and type has been reset. Please check carefully. viation from the protocol or treatment received. whereas in the noncooled group, 44 infants died Consistent with previous reports,10,11 neurologic and 42 had severe disability (Table 2) (relative risk JOB: 360xx ISSUE: xx-xx-09 outcomes are presented for survivors who had for either outcome, 0.86; 95% confidence interval available follow-up data. [CI], 0.68 to 1.07; P = 0.17). The result was materi- Comparative statistical analysis entailed calcu- ally unchanged when adjusted for severity of ab- lating the relative risks plus the 95% confidence normality on amplitude-integrated electroenceph- intervals for all dichotomous outcomes, the mean alography, sex, or age at randomization. differences plus 95% confidence intervals for nor- mally distributed, continuous outcomes (using Adverse Outcomes analysis of covariance where appropriate), and the The incidence of adverse events was similar in the median differences plus 95% confidence intervals two groups (Table 3). Hypotension, thrombocy- for skewed continuous variables. In addition, or- topenia, prolonged coagulation time, and intra- dered categorical variables were examined with the cranial hemorrhage (seen only on MRI) were fre- use of the chi-square test for trend. An adjusted quently observed in both groups. Serious adverse analysis of the primary outcome was performed events other than death were uncommon and were to investigate the effect of known prognostic fac- not associated with cooling. Two infants in the tors. All statistical tests were two-sided and were cooled group and one in the noncooled group had not adjusted for multiple comparisons. sinus thrombosis noted on MRI. Another infant Prespecified subgroup analyses were performed in the cooled group had a thrombus in the aorta,1352 n engl j med 361;14 nejm.org october 1, 2009 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • Moder ate Hypothermia for Perinatal Asphyxial Encephalopathy Table 1. Baseline Characteristics of the Infants.* Cooled Group Noncooled Group Characteristic (N = 163) (N = 162) P Value Male sex — no. (%) 101 (62) 88 (54) 0.16 Gestational age — wk Median 40.3 40.1 0.22 IQR 39.1–41.3 38.8–41.1 Birth weight — g Median 3450 3350 0.18 IQR 2957–3873 3044–3729 Head circumference — cm Median 35.0 35.0 0.53 IQR 34.0–36.0 34.0–35.9 Age at randomization Median — hr 4.7 4.7 0.88 IQR — hr 3.8–5.4 3.5–5.5 0 to <4 hr — no. (%) 48 (29) 57 (35) 0.27 4 to 6 hr — no. (%) 115 (71) 105 (65) Maternal pyrexia during labor — no. (%)† 10 (6) 10 (6) 0.94 Delivery complications — no. (%) 115 (71) 119 (74) 0.55 Apgar score at 10 min Median 4 4 0.15 IQR 2–5 2–5 <5 — no. (%) 110 (83) 105 (77) 0.21 Resuscitation required at 10 min of age — no. (%) 149 (91) 151 (93) 0.54 Clinical seizures — no. (%) 92 (56) 83 (51) 0.35 Temperature at randomization Mean ±SD — °C 36.6±1.1 36.5±1.2 0.24 <35.5°C — no. (%) 18 (11) 25 (16) 0.24 Abnormality on aEEG at randomization — no. (%) Moderate 65 (40) 67 (41) Severe 98 (60) 95 (59) 0.79* Data were unavailable for some patients: for gestational age, 16 patients in the cooled group and 17 in the noncooled group; for birth weight, 1 patient in the cooled group and 1 in the noncooled group; for head circumference, 41 pa- tients in the cooled group and 44 in the noncooled group; for maternal pyrexia, 2 patients in the cooled group and 6 in the noncooled group; for delivery complications, 2 patients in the cooled group and 2 in the noncooled group; for Apgar score at 10 minutes, 31 patients in the cooled group and 26 in the noncooled group; and for temperature at ran- domization, 2 patients in the noncooled group. The term aEEG denotes amplitude-integrated electroencephalography, and IQR interquartile range.† Pyrexia was defined as a temperature of 37.6°C or more.as well as an umbilical arterial catheter and a he- occurred after the withdrawal of care in 34 of thematocrit of 70%. No case of renal failure requir- 39 (87%) in the cooled group and 29 of the 39 (74%)ing dialysis occurred. in the noncooled group. Outcomes were significantly improved in theSecondary Outcomes at 18 Months cooled group with regard to 5 of the 12 second-The mortality rate was similar in both groups. ary neurologic outcomes assessed (Table 2). TheForty-two infants in the cooled group died, as did rate of survival without a neurologic abnormality44 infants in the noncooled group; in each group, was significantly increased in the cooled group39 of those died before hospital discharge. Death (71 of 163 infants [44%], vs. 45 of 162 [28%] in n engl j med 361;14 nejm.org october 1, 2009 1353 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • The n e w e ng l a n d j o u r na l of m e dic i n e However, the effect of cooling did not significant- 40 ly vary according to the severity of abnormality on amplitude-integrated electroencephalography 39 (P = 0.23 for interaction). The results were similar No cooling when the analysis was carried out with the results 38 of amplitude-integrated electroencephalography 37 classified as in the CoolCap study.10 The effect of treatment group did not vary significantly on the Mean Rectal Temperature (°C) 36 basis of time to randomization: among the 105 infants randomly assigned to a group less than 35 4 hours after birth, the relative risk for the primary outcome with cooling was 0.77 (95% CI, 0.44 to 34 1.04), whereas among the 220 remaining infants who were randomly assigned between 4 and 33 6 hours after birth, the relative risk was 0.95 Cooling (95% CI, 0.72 to 1.25; P = 0.21 for interaction). 32 31 Discussion 30 In this trial of near-term infants with perinatal asphyxia, we found no significant difference in the 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 risk of the primary outcome, the combined rates Hours since Randomization of death or severe disability, between the cooled group and the noncooled group. However, cooling Figure 2. Mean Rectal Temperatures during the Study, According to resulted in consistent improvement in second- Treatment Group. AUTHOR: Azzopardi ICM RETAKE 1st 2nd ary outcomes, including a significant increase in REG indicate 2 SDs. 2 The vertical bars F FIGURE: 2 of CASE 3rd Revised the rate of survival without neurologic abnormal- EMail Line 4-C SIZE ities and improved neurodevelopmental outcomes ARTIST: ts Enonthe noncooled group; H/T Combo relative H/T risk, 1.57; 95% CI, 22p3 among survivors. 1.16 to AUTHOR,= 0.003). Among survivors, cooling 2.12; P PLEASE NOTE: The primary outcome of TOBY, as in the Figure has been redrawn and type has been reset. palsy (relative resulted in reduced risks of cerebral CoolCap and NICHD trials, was a composite end risk, 0.67; 95% CI,carefully. 0.96; P = 0.03) and ab- Please check 0.47 to point, chosen because of concerns that cooling normal GMFCS score (relative risk, 0.63; 95% CI, JOB: 360xx ISSUE: xx-xx-09 might increase survival with additional disability. 0.45 to 0.89; P = 0.007) and resulted in improve- Results of all three trials are consistent with re- ments in the Mental Developmental Index and spect to this primary outcome, with point esti- Psychomotor Developmental Index scores (P = 0.03 mates supporting a benefit from hypothermia: the for each), and the GMFCS score (P = 0.01). The relative risk associated with cooling (vs. no cool- rate of multiple neurodevelopmental abnormali- ing) was 0.82 (95% CI, 0.66 to 1.02) in the CoolCap ties was 21 of 112 in the cooled group, as com- study, 0.72 (95% CI, 0.71 to 0.93) in the NICHD pared with 33 of 110 in the noncooled group trial (which included infants with moderate dis- (relative risk, 0.63; 95% CI, 0.39 to 1.01; P = 0.05). ability), and 0.86 (95% CI, 0.68 to 1.07) in the Results from the complete analysis of the neurode- present trial. velopmental assessments are provided in the Sup- Our categorization of neurologic outcomes is plementary Appendix. consistent with that used in the CoolCap and NICHD trials and previous systematic reviews,12-17 Subgroup Analyses facilitating the comparison of our findings with More infants with severely abnormal results on previous results. We found a significant increase amplitude-integrated electroencephalography at in the rate of survival without neurologic abnor- the time of randomization died or had a severe mality with cooling (relative risk, 1.57; 95% CI, disability than did those with moderately abnor- 1.16 to 2.12); the NICHD and CoolCap trials, mal results (109 of 193 [56%] vs. 50 of 132 [38%]; which were smaller than the present trial, showed relative risk, 1.49; 95% CI, 1.16 to 1.91; P = 0.001). nonsignificant benefits with regard to this out-1354 n engl j med 361;14 nejm.org october 1, 2009 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • Moder ate Hypothermia for Perinatal Asphyxial Encephalopathy Table 2. Main Neurodevelopmental Outcomes at 18 Months. Relative Risk Outcome Cooled Group Noncooled Group P Value (95% CI) no./total no. (%) Primary outcome Combined death and severe 74/163 (45) 86/162 (53) 0.17 0.86 (0.68–1.07) neurodevelopmental disability Secondary outcomes* Death 42/163 (26) 44/162 (27) 0.78 0.95 (0.66–1.36) Severe neurodevelopmental disability 32/120 (27) 42/117 (36) 0.13 0.74 (0.51–1.09) Survival without neurologic abnormality 71/163 (44) 45/162 (28) 0.003 1.57 (1.16–2.12) Multiple neurodevelopmental disabilities 21/112 (19) 33/110 (30) 0.05 0.63 (0.39–1.01) BSID-II Mental Developmental Index score 0.03 for trend <70 28/115 (24) 38/110 (35) 0.09 0.70 (0.47–1.06) 70–84 6/115 (5) 12/110 (11) ≥85 81/115 (70) 60/110 (55) 0.01 1.29 (1.05–1.59) BSID-II Psychomotor Developmental Index 0.03 for trend score <70 27/114 (24) 37/109 (34) 0.09 0.70 (0.46–1.06) 70–84 9/114 (8) 14/109 (13) ≥85 78/114 (68) 58/109 (53) 0.02 1.29 (1.04–1.60) GMFCS score 0.01 for trend No abnormality 85/120 (71) 63/117 (54) 0.007 1.32 (1.07–1.61) 1–2 11/120 (9) 18/117 (15) 3–5 24/120 (20) 36/117 (31) 0.06 0.65 (0.41–1.02) Cerebral palsy 33/120 (28) 48/117 (41) 0.03 0.67 (0.47–0.96) Hearing loss not corrected by aids 4/114 (4) 7/108 (6) 0.31 0.54 (0.16–1.80) No useful vision 8/119 (7) 12/114 (11) 0.30 0.64 (0.27–1.50) Seizures requiring anticonvulsant agents at 12/116 (10) 16/116 (14) 0.42 0.75 (0.37–1.51) time of assessment Head circumference at follow-up >2 SD 24/114 (21) 28/112 (25) 0.48 0.84 (0.52–1.36) below the mean* Scores on the Bayley Scales of Infant Development II (BSID-II) are assessed relative to a standardization mean (±SD) of 100±15, with higher scores indicating better performance. Scores on the Gross Motor Function Classification System (GMFCS) can range from 1 to 5, with higher scores indicating greater impairment. CI denotes confidence interval.come but had similar point estimates. The relative TOBY and the CoolCap trial showed that the in-risk in the NICHD trial was 1.51 (95% CI, 0.94 creased risk of death or severe disability in infantsto 2.42),23 and the relative risk in the CoolCap with the most abnormal grade on amplitude-trial was 1.48 (95% CI, 0.89 to 2.45) (Gunn A, integrated electroencephalography was unaffect-University of Auckland, New Zealand: personal ed by cooling, but the CoolCap results suggestedcommunication). a reduction of the risk in the subgroup with less Although there is striking homogeneity among severely abnormal findings.results of these three trials, there are also some These discrepancies among results may be ex-differences. Only the NICHD trial detected a sig- plained in part by differences in the trial proto-nificant effect of hypothermia on the primary cols. In all three trials, the whole-body tempera-outcome, and only TOBY detected significant im- ture (as measured in the rectum or esophagus)provements in specific neurologic outcomes. Both was reduced, but the strategies for cooling and n engl j med 361;14 nejm.org october 1, 2009 1355 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • The n e w e ng l a n d j o u r na l of m e dic i n e Table 3. Adverse Outcomes, According to Treatment Group.* Relative Risk or Cooled Group Noncooled Group Median Difference Outcome (N = 163) (N = 162) P Value (95% CI)† Total duration of hospital care — days Median 12 13 0.13 1 (0–4) IQR 8–18 9–25 Persistent hypotension — no./total no. (%)‡ 126/163 (77) 134/162 (83) 0.22 0.93 (0.84–1.04) Prolonged coagulation time — no./total no. (%) 67/163 (41) 72/161 (45) 0.51 0.92 (0.71–1.18) Thrombocytopenia — no./total no. (%) 94/163 (58) 80/161 (50) 0.15 1.16 (0.95–1.42) Intracranial hemorrhage — no./total no. (%)§ 25/64 (39) 21/67 (31) 0.35 1.25 (0.78–1.99) Pulmonary diagnoses — no./total no. (%) Pneumonia 5/163 (3) 5/162 (3) 0.99 0.99 (0.29–3.37) Pulmonary air leak 9/163 (6) 3/162 (2) 0.08 2.98 (0.82–10.80) Pulmonary hemorrhage 5/163 (3) 3/162 (2) 0.48 1.66 (0.40–6.82) Pulmonary hypertension 16/163 (10) 9/162 (6) 0.15 1.77 (0.80–3.88) Necrotizing enterocolitis — no./total no. (%) 1/163 (<1) 0/162 Cardiac arrhythmia — no./total no. (%)¶ 8/163 (5) 3/162 (2) 0.13 2.65 (0.72–9.81) Culture-proven sepsis — no./total no. (%) 20/163 (12) 20/162 (12) 0.98 0.99 (0.56–1.78) * No case of renal failure requiring dialysis occurred. CI denotes confidence interval, and IQR interquartile range. † Values are relative risks except for total duration of hospital care, for which the median difference is shown. ‡ Hypotension was defined as a mean blood pressure of 40 mm Hg or less. § Intracranial hemorrhage was identified on magnetic resonance imaging (MRI); 39 of the 46 cases were subdural (10 moderate and 29 mild). No intracranial hemorrhage was identified on cranial ultrasonography. Two cases of sinus thrombosis in the cooling group, and one case in the noncooled group, were also noted on MRI. ¶ All but three cases of cardiac arrhythmia consisted of sinus bradycardia of less than 80 beats per minute. The other three cases consisted of ventricular arrhythmia; two of these occurred in the noncooled group. the target temperatures varied: temperature was hypothermia on mortality rate in the NICHD study decreased to 33 to 34°C with the use of cooling as compared with TOBY. blankets in TOBY and the NICHD trial and to Elevation of body temperature to greater than 34 to 35°C by means of scalp cooling in the 38°C was observed in several noncooled infants CoolCap study. The NICHD trial recorded slight- in the NICHD and CoolCap trials and was associ- ly higher temperatures in the control group than ated with a worse outcome in the CoolCap trial.11,24 did the other two trials. In TOBY, but not the A rectal temperature of more than 38°C was also other two trials, cooling was initiated during noted in some noncooled infants in TOBY. Experi- transport to the treatment center. In TOBY and mental data showing that pyrexia may adversely the CoolCap trial, but not the NICHD trial, pa- affect neurodevelopment support the possibility tients were selected on the basis of the presence that increased temperatures may contribute to the of abnormalities on amplitude-integrated electro- poorer outcomes seen in the noncooled groups; encephalography in addition to clinical criteria. however, it is also possible that the relationship Differences in local practices for withdrawal of between higher elevation of body temperature and care may also have affected outcomes. Withdrawal poor outcome reflects reverse causation (i.e., as- was slightly more common in the control group phyxia resulting in impairment of temperature than in the cooled group in the NICHD study but regulation). was more common in the cooled group than in Consistent with findings in the earlier trials, the control group in TOBY; these results may par- in our study, minor respiratory and cardiovascular tially account for the greater apparent effect of events were common, but serious adverse events1356 n engl j med 361;14 nejm.org october 1, 2009 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • Moder ate Hypothermia for Perinatal Asphyxial Encephalopathywere rare and were not associated with cooling. havior and learning, fine motor development, at-Mild-to-moderate intracranial hemorrhage that tention, and psychosocial health.26was not visible on cranial ultrasonography was In conclusion, TOBY did not show a significantfrequently seen on MRI in both groups, and sinus reduction in the combined rates of death and se-thrombosis occurred very infrequently in both vere disability with cooling, as compared with nogroups. cooling, but did show a significant improvement No trial, to our knowledge, has yet reported in several secondary neurologic outcomes amongneurologic outcomes at ages older than 18 months. survivors. Whether this improvement is main-Neurodevelopmental assessments at 18 months tained in the longer term needs to be ascertained.may not reliably predict later outcomes.25 Althoughit is likely that severe neuromotor disability will Supported by grants from the U.K. Medical Research Council and the U.K. Department of Health.have been correctly identified at 18 months, less No potential conflict of interest relevant to this article was re-severe impairments are not reliably assessable at ported.this age. Assessment later in childhood (e.g., at We thank the Imperial College Healthcare Biomedical Research Centre and Bliss, the Special Care Baby Charity, for their advice6 to 7 years of age) is necessary for accurate, com- and support, as well as all the parents and infants who took partprehensive evaluation of cognitive function, be- in the study. AppendixMembers of the TOBY Study Group are as follows: Project Management Group — D. Azzopardi (chief investigator), P. Brocklehurst(chief investigator), A.D. Edwards (principal investigator), H. Halliday (principal investigator), M. Levene (principal investigator), M.Thoresen (principal investigator), A. Whitelaw (principal investigator), S. Ayers (National Perinatal Epidemiology Unit [NPEU] informa-tion technology coordinator), U. Bowler (NPEU Clinical Trials Unit [CTU] senior trials manager), M. Gallagher (NPEU data manager),E. Juszczak (NPEU CTU head of trials), C. Mulhall (NPEU TOBY study coordinator), B. Strohm (NPEU TOBY research nurse and studycoordinator); Writing Committee — D. Azzopardi (chief investigator), P. Brocklehurst (chief investigator), A.D. Edwards (principal in-vestigator), E. Juszczak (NPEU CTU head of trials); Trial Steering Committee — N. McIntosh (chair), Child Life and Health, Universityof Edinburgh, Edinburgh, United Kingdom (UK); D. Azzopardi, Imperial College London, London; H. Baumer, Derriford Hospital,Plymouth, UK; P. Brocklehurst, NPEU, University of Oxford, Oxford, UK; C. Doré, Medical Research Council (MRC) CTU; D. Elbourne,London School of Hygiene and Tropical Medicine, London; R. Parnell, Scope Data Monitoring and Ethics Committee; R. Cooke (chair),Liverpool Women’s Hospital, University of Liverpool, Liverpool, UK; H. Davies, National Research Ethics Service; A. Johnson, Univer-sity of Oxford, Oxford, UK; S. Richmond, Sunderland District General Hospital, University of Newcastle, Newcastle, UK; P. Yudkin,Division of Public Health and Primary Health Care, University of Oxford, Oxford, UK; Trial Statisticians (NPEU) — S. Gates, EdmundJuszczak, M. Quigley; Trial Health Economists (NPEU) — O. Eddama, J. Henderson, S. Petrou; Clinical Research Fellow — O. Kapellou;Follow-up Pediatricians — L. Dyet, E. Porter, Imperial College London, London; G. Mero, Jósa András County Hospital, Nyíregyháza,Hungary; B. Vollmer, Karolinska Institutet, Stockholm; E. Goldstein, Soroka Medical Center, Beersheeva, Israel; Specialist Adviser — B.Hutchon, Royal Free Hospital, London; Cranial Ultrasonography Interpretation — C. Hagmann, University College London, London;Bayley Scales of Infant Development II (BSID-II) Training — S. Johnson, University of Nottingham, Nottingham, UK; MRI Evaluation— L. Ramenghi, University of Milan, Milan; M. Rutherford, MRC Clinical Sciences Centre, Imperial College London, London; Centersfor Recruitment and Data Collection for MRI Evaluation (in descending order of no. of infants recruited, in parentheses) — Hammer-smith Hospital, London (54) — D. Azzopardi, A.D. Edwards, O. Kapellou, P. Corcoran; First Department of Pediatrics, SemmelweisUniversity Hospital, Budapest, Hungary (24) — M. Szabó, A. Róka, E. Bodrogi; Homerton Hospital, London (20) — E. Maalouf, C.Harris; Southmead Hospital, Bristol, UK (20) — A. Whitelaw, S. Lamburne; University College Hospital, London (19) — N. Robertson,A. Kapetanakis; St. George’s Hospital, London (18) — K. Farrer, L. Kay-Smith; Royal Maternity Hospital, Belfast, UK (17) — H. Hal-liday, D. Sweet; Liverpool Women’s Hospital, Liverpool, UK (15) — M. Weindling, A.S. Burke; St. Michael’s Hospital, Bristol, UK (14)— M. Thoresen, J. Tooley, J. Kemp; Leicester Royal Infirmary, Leicester, UK (12) — A. Currie, M. Hubbard; Royal Sussex County Hos-pital, Brighton, UK (10) — P. Amess; Queen Silvia’s Hospital, Gothenburg, Sweden (9) — K. Thiringer, A. Flisberg; Leeds GeneralInfirmary, Leeds, UK (8) — M. Levene, A. Harrop; Nottingham City Hospital, Nottingham, UK (8) — S. Watkin, D. Jayasinghe; JohnRadcliffe Hospital, Oxford, UK (7) — E. Adams; Karolinska Institutet, Stockholm (6) — C. Lothian, M. Blennow; Medway MaritimeHospital, Gillingham, UK (6) — S. Rahman, B. Jani, K. Vandertak; Luton and Dunstable Hospital, Luton, UK (5) — S. Skinner, Y. Mil-lar; Queen’s Medical Centre, Nottingham, UK (5) — N. Marlow, S. Wardle; Jessop Wing, Sheffield, UK (4) — M. Smith; Royal VictoriaInfirmary, Newcastle, UK (4) — J. Berrington; Soroka Medical Center, Beersheva, Israel (4) — K. Marks; Bradford Royal Infirmary,Bradford, UK (3) — S. Chatfield; Heartlands Hospital, Birmingham, UK (3) — S. Rose; New Cross Hospital, Wolverhampton, UK (3)— B. Kumararatne, L. Greig; Norfolk and Norwich University Hospital, Norwich, UK (3) — P. Clarke; Lund University Hospital, Lund,Sweden (3) — V. Fellman; Wishaw General Hospital, Wishaw, UK (3) — R. Abara; City Hospital, Birmingham, UK (2) — D. Armstrong;Erinville Hospital–Cork University Hospital, Cork, Ireland (2) — D. Murray; Hospital for Children and Adolescents, Helsinki (2) — M.Metsaranta; Queen Mother’s Hospital, Glasgow, UK (2) — J. Simpson; Singleton Hospital, Swansea, UK (2) — J. Matthes; SouthernGeneral Hospital, Glasgow, UK (2) — P. MacDonald; University Hospital of Wales, Cardiff, UK (2) — S. Cherian; Princess Royal Ma-ternity Hospital, Glasgow, UK (1) — L. Jackson; Royal Cornwall Hospital, Truro, UK (1) — P. Munyard; Royal Devon and ExeterFoundation Trust, Exeter, UK (1) — M. Quinn; St. Mary’s Hospital, Manchester, UK (1) — S. Mitchell; James Cook University Hospital,Middlesbrough, UK (0) — S. Sinha; Derriford Hospital, Plymouth, UK (0) — J. Eason; Department of Pediatrics, Oulu University Hos-pital, Oulu, Finland (0) — M. Hallman. n engl j med 361;14 nejm.org october 1, 2009 1357 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • Moder ate Hypothermia for Perinatal Asphyxial Encephalopathy References 1. Thoresen M, Penrice J, Lorek A, et al. et al. Moderate hypothermia in neonatal troencephalography. Pediatrics 1999;103: Mild hypothermia after severe transient encephalopathy: safety outcomes. Pediatr 1263-71. hypoxia-ischemia ameliorates delayed ce- Neurol 2005;32:18-24. 19. Allmark P, Mason S. Improving the rebral energy failure in the newborn pig- 10. Gluckman PD, Wyatt JS, Azzopardi D, quality of consent to randomised con- let. Pediatr Res 1995;37:667-70. et al. Selective head cooling with mild sys- trolled trials by using continuous consent 2. Sirimanne ES, Blumberg RM, Bossa- temic hypothermia after neonatal enceph- and clinician training in the consent pro- no D, et al. The effect of prolonged modi- alopathy: multicentre randomised trial. cess. J Med Ethics 2006;32:439-43. fication of cerebral temperature on outcome Lancet 2005;365:663-70. 20. Palisano RJ, Hanna SE, Rosenbaum after hypoxic-ischemic brain injury in the 11. Shankaran S, Laptook AR, Ehrenkranz PL, et al. Validation of a model of gross infant rat. Pediatr Res 1996;39:591-7. RA, et al. Whole-body hypothermia for motor function for children with cerebral 3. Amess PN, Penrice J, Cady EB, et al. neonates with hypoxic-ischemic enceph- palsy. Phys Ther 2000;80:974-85. Mild hypothermia after severe transient alopathy. N Engl J Med 2005;353:1574-84. 21. Haataja L, Mercuri E, Regev R, et al. hypoxia-ischemia reduces the delayed rise 12. Shah PS, Ohlsson A, Perlman M. Hy- Optimality score for the neurologic exami- in cerebral lactate in the newborn piglet. pothermia to treat neonatal hypoxic ische- nation of the infant at 12 and 18 months of Pediatr Res 1997;41:803-8. mic encephalopathy: systematic review. age. J Pediatr 1999;135:153-61. 4. Edwards AD, Yue X, Squier MV, et al. Arch Pediatr Adolesc Med 2007;161:951-8. 22. Bayley N. Bayley scales of infant devel- Specific inhibition of apoptosis after cere- 13. Schulzke SM, Rao S, Patole SK. A sys- opment. 2nd ed. San Antonio, TX: Psy- bral hypoxia-ischaemia by moderate post- tematic review of cooling for neuropro- chological Corporation, 1993. insult hypothermia. Biochem Biophys Res tection in neonates with hypoxic ischemic 23. Shankaran S, Pappas A, Laptook AR, Commun 1995;217:1193-9. encephalopathy — are we there yet? BMC et al. Outcomes of safety and effective- 5. Bona E, Hagberg H, Loberg EM, Bå- Pediatr 2007;7:30. ness in a multicenter randomized, con- genholm R, Thoresen M. Protective effects 14. Jacobs S, Hunt R, Tarnow-Mordi W, trolled trial of whole-body hypothermia of moderate hypothermia after neonatal Inder T, Davis P. Cooling for newborns for neonatal hypoxic-ischemic encepha- hypoxia-ischemia: short- and long-term with hypoxic ischaemic encephalopathy. lopathy. Pediatrics 2008;122(4):e791-e798. outcome. Pediatr Res 1998;43:738-45. Cochrane Database Syst Rev 2007;4: 24. Wyatt JS, Gluckman PD, Liu PY, et al. 6. Colbourne F, Corbett D, Zhao Z, Yang CD003311. Determinants of outcomes after head J, Buchan AM. Prolonged but delayed post- 15. Higgins RD, Raju TN, Perlman J, et al. cooling for neonatal encephalopathy. Pe- ischemic hypothermia: a long-term out- Hypothermia and perinatal asphyxia: ex- diatrics 2007;119:912-21. come study in the rat middle cerebral ar- ecutive summary of the National Institute 25. Barnett AL, Guzzetta A, Mercuri E, et tery occlusion model. J Cereb Blood Flow of Child Health and Human Development al. Can the Griffiths scales predict neuro- Metab 2000;20:1702-8. workshop. J Pediatr 2006;148:170-5. motor and perceptual-motor impairment 7. Gunn AJ, Gluckman PD, Gunn TR. 16. Barks JD. Current controversies in hy- in term infants with neonatal encepha- Selective head cooling in newborn infants pothermic neuroprotection. Semin Fetal lopathy? Arch Dis Child 2004;89:637-43. after perinatal asphyxia: a safety study. Neonatal Med 2008;13:30-4. 26. Voss W, Neubauer AP, Wachtendorf M, Pediatrics 1998;102:885-92. 17. Kirpalani H, Barks J, Thorlund K, Verhey JF, Kattner E. Neurodevelopmental 8. Azzopardi D, Robertson NJ, Cowan Guyatt G. Cooling for neonatal hypoxic outcome in extremely low birth weight in- FM, Rutherford MA, Rampling M, Edwards ischemic encephalopathy: do we have the fants: what is the minimum age for reliable AD. Pilot study of treatment with whole answer? Pediatrics 2007;120:1126-30. developmental prognosis? Acta Paediatr body hypothermia for neonatal encepha- 18. al Naqeeb N, Edwards AD, Cowan FM, 2007;96:342-7. lopathy. Pediatrics 2000;106:684-94. Azzopardi D. Assessment of neonatal en- Copyright © 2009 Massachusetts Medical Society. 9. Eicher DJ, Wagner CL, Katikaneni LP, cephalopathy by amplitude-integrated elec- powerpoint slides of journal figures and tables At the Journal’s Web site, subscribers can automatically create PowerPoint slides. In a figure or table in the full-text version of any article at NEJM.org, click on Get PowerPoint Slide. A PowerPoint slide containing the image, with its title and reference citation, can then be downloaded and saved.1358 n engl j med 361;14 nejm.org october 1, 2009 Downloaded from www.nejm.org at UNIVERSITY OF WASHINGTON on December 10, 2009 . Copyright © 2009 Massachusetts Medical Society. All rights reserved.
    • ArticlesLong-term risk of epilepsy after traumatic brain injury inchildren and young adults: a population-basedcohort studyJakob Christensen, Marianne G Pedersen, Carsten B Pedersen, Per Sidenius, Jørn Olsen, Mogens VestergaardSummaryBackground The risk of epilepsy shortly after traumatic brain injury is high, but how long this high risk lasts is Lancet 2009; 373: 1105–10unknown. We aimed to assess the risk of epilepsy up to 10 years or longer after traumatic brain injury, taking into Published Onlineaccount sex, age, severity, and family history. February 23, 2009 DOI:10.1016/S0140- 6736(09)60214-2Methods We identified 1 605 216 people born in Denmark (1977–2002) from the Civil Registration System. We obtained See Comment page 1060information on traumatic brain injury and epilepsy from the National Hospital Register and estimated relative risks Department of Neurology,(RR) with Poisson analyses. Aarhus University Hospital, Aarhus, DenmarkFindings Risk of epilepsy was increased after a mild brain injury (RR 2·22, 95% CI 2·07–2·38), severe brain injury (J Christensen MD,(7·40, 6·16–8·89), and skull fracture (2·17, 1·73–2·71). The risk was increased more than 10 years after mild brain P Sidenius MD); Department of Clinical Pharmacologyinjury (1·51, 1·24–1·85), severe brain injury (4·29, 2·04–9·00), and skull fracture (2·06, 1·37–3·11). RR increased (J Christensen) and Nationalwith age at mild and severe injury and was especially high among people older than 15 years of age with mild (3·51, Centre for Register-based2·90–4·26) and severe (12·24, 8·52–17·57) injury. The risk was slightly higher in women (2·49, 2·25–2·76) than in Research (M G Pedersen MSc,men (2·01, 1·83–2·22). Patients with a family history of epilepsy had a notably high risk of epilepsy after mild (5·75, C B Pedersen MSc), University of Aarhus, Denmark; Southern4·56–7·27) and severe brain injury (10·09, 4·20–24·26). California Injury Prevention Research Centre (SCIPRC),Interpretation The longlasting high risk of epilepsy after brain injury might provide a window for prevention of School of Public Health, UCLA, CA, USA (J Olsen MD),post-traumatic epilepsy. Department of Epidemiology (J Olsen) and Department ofFunding Danish Research Agency, P A Messerschmidt and Wife’s Foundation, Mrs Grethe Bønnelycke’s Foundation. General Practice (M Vestergaard MD), Institute of Public Health, University ofIntroduction Methods Aarhus, Aarhus, DenmarkTraumatic brain injury raises the risk of epilepsy,1 but Study population Correspondence to:little is known about the duration of the increased risk We used data from the Danish Civil Registration System Dr Jakob Christensen,and the factors that modify the risk, especially in children (CRS)8 to identify all people born in Denmark between Department of Neurology,and young adults.2 In hospital-based case series, the risk Jan 1, 1977, and Dec 31, 2002. All liveborn children and Aarhus University Hospital, Norrebrogade 44,of epilepsy 1–2 years after moderate to severe brain injury new residents in Denmark are assigned a unique DK-8000 Aarhus C, Denmarkis related to some CT or MRI findings and is high in personal identification number (CRS number) together jakob@farm.au.dkpeople who had had neurosurgical procedures.2–6 In a with information on vital status, emigration frompopulation-based study, age in people who had traumatic Denmark, and CRS numbers of mothers, fathers, andbrain injury at age 65 years or older and time since and siblings. The CRS number links individual informationseverity of injury were significant risk factors for epilepsy,1 in all national registries and provides identification ofbut only a few studies have included children and young family members and links parents with their children.adults.1–4 In some of these studies,2,4 acute seizures in the Identity of individuals in the study was blinded to thefirst week after brain injury were associated with a high investigators, and the study did not involve contact withrisk of epilepsy. Studies of epilepsy related to level of individual patients. The study therefore did not needconsciousness (eg, assessed with the Glasgow coma approval from the ethics committee according to Danishscale) and duration of post-traumatic amnesia after brain laws, but the project was approved by the Danish Datainjury have given conflicting results.1,3,4 Protection Agency. No effective prophylaxis for epilepsy after traumaticbrain injury is available, and trials with preventive drug Data collectionhave been discouraging.7 However, better information Information about brain injury and epilepsy was obtainedabout prognostic factors might help the development of from the Danish National Hospital Register,9 whichnew prevention strategies and treatment.5 contains information on all discharges from Danish We studied the risk of epilepsy in a large hospitals since 1977; outpatients have been included in thepopulation-based cohort of children and young adults register since 1995. All treatment is free of charge forand considered time since injury, sex, age, severity, and Danish residents. Patients admitted to the only privatefamily history of epilepsy. epilepsy hospital in Denmark are also recorded in thewww.thelancet.com Vol 373 March 28, 2009 1105
    • Articles Danish National Hospital Register. Specialists in neurology Patients diagnosed New cases (per Adjusted relative risk p value with epilepsy 1000 person-years) (95% CI) working in private outpatient clinics also treat patients with epilepsy, but these contacts are not recorded in the Time (years) since mild brain injury Danish National Hospital Register. 0·0–0·5 162 4·67 5·46 (4·67–6·37) <0·0001 Diagnostic information in the National Hospital 0·5–1·0 78 2·37 2·91 (2·33–3·64) <0·0001 Register is based on the International Classification of 1·0–2·0 109 1·78 2·26 (1·87–2·73) <0·0001 Diseases, 8th revision (ICD-8) from 1977–93, and ICD-10 2·0–3·0 99 1·79 2·33 (1·91–2·84) <0·0001 from 1994–2002. 3·0–5·0 138 1·50 1·99 (1·68–2·36) <0·0001 Cohort members, their parents and siblings were 5·0–10·0 154 1·14 1·56 (1·33–1·83) <0·0001 classified with epilepsy if they had been hospitalised or in ≥10·0 97 1·00 1·51 (1·24–1·85) <0·0001 outpatient care with a diagnosis of epilepsy (ICD-8: 345; No mild injury 16 633 0·87 1·00 ·· ICD-10: G40,G41).10–13 By use of the CRS numbers, we Time (years) since severe brain injury linked parents and siblings registered with an epilepsy 0·0–0·5 35 19·62 21·26 (15·25 to 29·62) <0·0001 diagnosis in the National Hospital Register. A person was 0·5–1·0 19 11·52 13·45 (8·57 to 21·09) <0·0001 recorded as having a family history of epilepsy if the date 1·0–2·0 18 6·06 7·42 (4·68 to 11·79) <0·0001 of first epilepsy diagnosis in a parent or sibling preceded 2·0–3·0 11 4·26 5·40 (2·99 to 9·76) <0·0001 their epilepsy diagnosis. 3·0–5·0 11 2·69 3·52 (1·95 to 6·35) <0·0001 Cohort members were classified with mild brain injury 5·0–10·0 15 3·22 4·40 (2·65 to 7·30) <0·0001 (concussion: ICD-8 850.99; ICD-10 S06.0), severe brain ≥10·0 7 2·94 4·29 (2·04 to 9·00) 0·0001 injury (structural brain injury: ICD-8 851.29-854.99; No severe injury 17 354 0·89 1·00 ·· ICD-10 S06.1-S06.9), or skull fracture (ICD-8 800.99–801.09, Time (years) since skull fracture 803.99; ICD-10: S02–S02.1, S02.7, S02.9), respectively, if 0·0–0·5 6 2·90 2·96 (1·33 to 6·60) 0·0078 they had been admitted or been in outpatient care with the 0·5–1·0 6 2·99 3·51 (1·58 to 7·83) 0·0021 relevant diagnosis.14,15 Time of onset of epilepsy and brain 1·0–2·0 13 3·38 4·30 (2·50 to 7·41) <0·0001 injury was defined as the first day of the first contact to the 2·0–3·0 5 1·39 1·81 (0·75 to 4·35) 0·1845 hospital with the relevant diagnosis. 3·0–5·0 9 1·36 1·78 (0·93 to 3·42) 0·0838 The definition of mild brain injury (concussion) in 5·0–10·0 16 1·21 1·55 (0·95 to 2·54) 0·0781 Denmark is based on the definition given by the American ≥10·0 23 1·46 2·06 (1·37 to 3·11) 0·0005 Congress of Rehabilitation Medicine.16 The diagnostic No fracture 17 392 0·89 1·00 ·· criteria include a relevant direct trauma against the head manifesting with changed brain function (ie, loss of Each form of injury led to a significant (p<0·0001) increase in risk of epilepsy relative to people without brain injury. consciousness, amnesia, confusion/disorientation, or Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brain injury was modified by time since first admission with brain injury for mild (p<0·0001) and severe (p<0·0001) brain focal [temporary] neurological deficit). Severity of mild injury but not skull fracture (p=0·16). brain injury should not include loss of consciousness longer than 30 min, a Glasgow coma scale of 13 or less Table 1: Time since first admission with brain injury and relative risk (RR) of epilepsy after 30 min, or post-traumatic amnesia longer than 24 h.17 Severe brain injury (structural brain injury) includes 35 Mild brain injury brain contusion or intracranial haemorrhage. Skull Severe brain injury Skull fracture fracture liable to be associated with disruption of brain 30 Reference function can occur alone or be associated with other types of brain injury and usually requires verification with a 25 radiograph or CT. Brain injuries recorded in the same patient within 14 days were categorised as the same event Relative risk of epilepsy according to the hierarchy of brain injury—severe brain 20 injury, skull fracture, and mild brain injury. For each type of brain injury, we calculated the age at first brain injury 15 (0–5, 5–10, 10–15, and ≥15 years), the length of first admission (0, 1–6, 7–13, 14–27, and ≥28 days), and time 10 since first brain injury (0–6 months, 6 months to 1 year, 1–2, 2–3, 3–5, 5–10, and ≥10 years). 5 Statistical analyses People were followed from birth until onset of epilepsy, 0 death, emigration from Denmark, or Dec 31, 2002, 0 1 2 3 4 5 6 7 8 9 ≥10 whichever came first. The incidence rate ratio (for these Years after injury analyses a good approximation of the relative risk, theFigure: Relative risk of epilepsy after brain injury in Denmark (1977–2002) term used in this Article) of epilepsy was estimated by1106 www.thelancet.com Vol 373 March 28, 2009
    • Articleslog-linear Poisson regression18 with the GENMOD Number of patients New cases (per Adjusted relative risk p valueprocedure in SAS (version 8.1). Because incidence of with epilepsy* 1000 person-years) (95% CI)epilepsy depends on age, sex, and calendar year,10 all the Age (years) at mild brain injuryrelative risks were adjusted for these factors. Age, 0–5 365 1·64 2·06 (1·86–2·29) <0·0001calendar year, age at first brain injury, duration of first 5–10 243 1·56 2·12 (1·87–2·41) <0·0001admission with brain injury, time since first brain injury, 10–15 117 1·54 2·25 (1·88–2·71) <0·0001and history of epilepsy in a parent or sibling were timedependent variables;19 all other variables were treated as ≥15 112 2·03 3·51 (2·90–4·26) <0·0001time independent. Age was categorised in quarter year No mild injury 16 633 0·87 1·00 ··age levels from birth to the first birthday, in 1 year age Age (years) at severe brain injurylevels from the first birthday to the 20th birthday, and as 0–5 51 6·26 7·20 (5·47–9·48) <0·000120–21 years and ≥22 years. Calendar year was categorised 5–10 24 4·96 6·18 (4·14–9·23) <0·0001in 1 year periods from 1977 to 2002. Likelihood ratio tests 10–15 11 3·56 4·91 (2·72–8·87) <0·0001were used to calculate p values and 95% CIs were ≥15 years 30 7·47 12·24 (8·52–17·57) <0·0001calculated by use of Wald’s test.19 The adjusted-score test20 No severe injury 17 354 0·89 1·00 ··suggested that the regression models were not subject to Age (years) at skull fractureoverdispersion. 0–5 52 1·53 1·95 (1·49–2·56) <0·0001 5–10 17 2·12 2·86 (1·78–4·60) <0·0001Role of the funding source 10–15 5 1·81 2·55 (1·06–6·12) 0·0368The sponsors had no role in the study design, data ≥15 years 4 1·71 2·75(1·03–7·34) 0·0433collection, data analysis, data interpretation, or writing of No skull fracture 17 392 0·89 1·00 ··the Article. All authors had full access to the data and Each form of injury led to a significant (p<0·0001) increase in risk of epilepsy relative to people without brain injury.approved the decision to submit the Article for publication Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brainin The Lancet. injury was modified by age at first admission with brain injury for mild (p<0·0001) and severe (p=0·02) brain injury but not skull fracture (p=0·55).Results Table 2: Age at first admission with brain injury and relative risk of epilepsyWe followed-up 1 605 216 for a total of19 527 337 person-years. During this study period,78 572 people had at least one traumatic brain injury, and (p=0·02) had a notably high risk of epilepsy (table 3). Forin the same period, 17 470 people developed epilepsy, of people with mild brain injury there was no associationwhom 1017 had a preceding brain injury. Follow-up was between duration of hospital stay and risk of epilepsystopped before the end of the study period for (p=0·73; table 3).45 677 people (2·9%) because of emigration from Table 4 shows the relative risk of epilepsy after brainDenmark (30 362 [1·9%]) or death (15 315 [1·0%]). injuries subdivided by family history of epilepsy. The Relative to no brain injury, the risk of epilepsy was two relative risk of epilepsy with a family history of thetimes higher after mild brain injury (RR 2·22, 95% CI disorder and mild brain injury is between what would2·07–2·38); seven times higher after severe brain injury have been predicted from a multiplicative model(7·40, 6·16–8·89); and two-times higher after skull (3·37×2·24=7·54) and from an additive modelfracture (2·17, 1·73–2·71). (3·37+2·24–1=4·61; table 4). The relative risk estimate Tables 1–3 show the risk of epilepsy after brain injury associated with severe brain injury and family history ofaccording to time since first admission with brain epilepsy of is almost the same as would have beeninjury, age at first brain injury, and duration of first predicted from an additive model (3·35+7·81–1=10·16;hospital stay with brain injury. table 4). We had very few people with epilepsy with skull The risk of epilepsy after mild (p<0·0001) and severe fracture and a family history of epilepsy (table 4).(p<0·0001) brain injury was highest during the first The relative risk of epilepsy after mild brain injury wasyears after injury, but remained high for more than higher among women (2·49, 2·25–2·76) than among10 years after the injury as compared with people without men (2·01, 1·83–2·22; p=0·003). There was nosuch a history (table 1, figure). For patients with skull interaction with sex for patients with skull fracturesfractures, risk of epilepsy did not vary significantly with (p=0·59) or severe brain injury (0·22). We calculated thetime since injury (p=0·16; table 1). risk of epilepsy for patients registered with brain injury Brain injury was associated with an increased risk of according to ICD-8 and ICD-10 (ie, patients diagnosed inepilepsy in all age groups (table 2). The risk increased the time period 1977 to 1993 and 1994 to 2002,with age for mild (p<0·0001) and severe (p=0·02) brain respectively). For patients with mild brain injury, the riskinjury and was highest among people older than of epilepsy was lower in the ICD-8 period (RR 1·89,15 years at injury. 1·71–2·10) than in the ICD-10 period (2·61, 2·37–2·87; Patients who had a long duration of hospital stay with p<0·0001). For severe brain injury, the risk of epilepsysevere brain injury (p<0·0001) and skull fracture was almost the same in the ICD-8 period (7·17,www.thelancet.com Vol 373 March 28, 2009 1107
    • Articles Discussion Number of patients New cases (per Adjusted relative risk# p value with epilepsy 1000 person-years) (95% CI) As previously shown in studies smaller than ours,1,2,6,21 risk of epilepsy increased after brain injury in relation to Hospital stay (days) for mild brain injury severity of brain injury. Risk was high for more than 0 256 1·73 2·22 (1·96–2·51) <0·0001 10 years after the brain injuries even for mild brain 1–6 563 1·60 2·20 (2·02–2·40) <0·0001 injury (concussion), a finding in contrast to that of a 7–13 9 2·08 3·01 (1·56–5·78) 0·0010 previous study showing no increased risk of epilepsy 14–27 4 2·54 3·68 (1·38–9·82) 0·0091 5 years after a mild brain injury.1 The discrepancy might ≥28 5 2·05 2·94 (1·22–7·07) 0·0159 result from different inclusion criteria for mild brain No mild injury 16 633 0·87 1·00 ·· injury and epilepsy,1,11 and an insufficient sample size to Hospital stay (days) for severe brain injury identify a moderate increase in risk.1 Our results suggest 0 12 1·73 2·09 (1·19–3·68) <0·0108 that time from brain injury to clinically overt symptoms 1–6 24 3·71 4·82 (3·23–7·20) <0·0001 (seizures) can span several years, leaving room for 7–13 15 7·04 9·42 (5·68–15·63) <0·0001 clinical intervention.5 However, animal studies suggest 14–27 18 13·11 18·01 (11·34–28·60) <0·0001 that a specific time window exists shortly after injury in ≥28 47 14·86 20·07 (15·06–26·74) <0·0001 which appropriate drugs might stop the epileptogenic No severe injury 17 354 0·89 1·00 ·· process,22 and antiepileptogenic trials after brain injury Hospital stay (days) for skull fracture in human beings have not shown drug treatment to be 0 8 2·13 2·72 (1·36–5·45) 0·0046 effective.7 In Denmark, seizure prophylaxis with 1–6 48 1·35 1·77 (1·33–2·35) <0·0001 antiepileptic drugs after brain injury was not used 7–13 10 1·99 2·70 (1·45–5·03) 0·0017 routinely in the study period.23 14–27 4 3·08 4·01 (1·51–10·69) 0·0055 We defined the onset of epilepsy as the first day of the ≥28 8 5·59 6·69 (3·35–13·38) <0·0001 first contact, although this is only an approximation. No fracture 17 392 0·89 1·00 ·· There may be a delay from the first seizure to diagnosis of epilepsy. We have previously validated the epilepsy Each form of injury led to a significant (p<0·0001) increase in risk of epilepsy relative to people without brain injury. Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brain diagnosis in a sample from the Danish National Hospital injury was modified by duration of first hospital stay with brain injury for severe brain injury (p<0·0001) and skull Register and found that 64% were registered in the fracture (p=0·02) but not mild brain injury (p=0·73). Danish National Hospital Register within 1 year of first Table 3: Duration of first hospital stay with brain injury and relative risk of epilepsy seizure, and 90% were registered within 5 years.11 Diagnostic delay might, therefore, explain part of the increased risk of epilepsy after the brain injury. Likewise, No family history of epilepsy Family history of epilepsy a delay between brain injury and diagnosis (eg, in Number of Adjusted relative p value Number of Adjusted relative p value patients with chronic subdural haematoma), could bias patients with risk (95% CI) patients with risk (95% CI) the estimates of epilepsy shortly after a brain injury epilepsy epilepsy diagnosis, but this effect is likely to be small, especially Mild brain injury in children. No 15 511 1·00 ·· 1122 3·37 (3·17–3·58) <0·0001 Brain injury might be the first presentation of epilepsy, Yes 766 2·24 (2·08–2·41) <0·0001 71 5·75 (4·56–7·27) <0·0001 in which the patient has a head trauma during an Severe brain injury unwitnessed seizure (reverse causation). In a No 16 166 1·00 ·· 1188 3·35 (3·16–3·56) <0·0001 subanalysis, we excluded patients diagnosed with Yes 11 7·81 (6·48–9·42) <0·0001 5 10·09 (4·20–24·26) <0·0001 epilepsy within the first 6 weeks of first brain injury Skull fracture diagnosis and found that the high risk of epilepsy No 16 202 1·00 ·· 1190 3·35 (3·16–3·55) <0·0001 remained for all types of brain injury, albeit in an Yes 75 2·28 (1·81–2·86) <0·0001 3 2·71 (0·87–8·41) 0·0842 attenuated form (data not shown). Although patients with infrequent seizures might remain undiagnosed Any brain injury more than 6 weeks, this problem probably affects only a No 15 338 1·00 ·· 1115 3·39 (3·19–3·61) <0·0001 small part of the delayed association between brain Yes 939 2·47 (2·31–2·65) <0·0001 78 5·73 (4·58–7·16) <0·0001 injury and epilepsy. The risk of epilepsy increased Patients might have been exposed to more than one type of brain injury at separate admissions/outpatient visits. slightly with age at time of mild brain injury, and was Relative risk adjusted for age and its interaction with sex and calendar year. highest for people over 15 years of age, indicating that Table 4: Family history and relative risk of epilepsy after traumatic brain injury susceptibility to epilepsy after brain injury increases with age. This finding is in line with results of a previous study identifying people aged 65 or more as being at 5·19–9·91) and the ICD-10 period (7·51, 6·02–9·38; high risk of epilepsy after brain injury.1 Alternatively, the p=0·82). For skull fracture, the risks of epilepsy were severity of brain injuries might increase with age, or comparable in the ICD-8 period (2·00, 1·54–2·58) and doctors might be more likely to hospitalise younger ICD-10 period (2·87, 1·85–4·46; p=0·17). children with less severe brain injuries, resulting in a1108 www.thelancet.com Vol 373 March 28, 2009
    • Articleslow relative risk of post-traumatic epilepsy in young age patients with and without brain injury, which we findgroups. unlikely. Post-traumatic epilepsy is thought to be typical of The Danish Hospital Register does not capture allsymptomatic epilepsy (ie, determined by environmental patients with epilepsy, because some outpatients mightfactors). However, twin studies suggest that genetic be treated in private practice. However, estimates offactors also play a part in localisation-related epilepsies, incidence (68·8 per 100 000 people per year) andmost of which are thought to be symptomatic or prevalence (0·6%) of epilepsy in Denmark based onprobably symptomatic.24 Family history of epilepsy and data from the Danish National Hospital Register weremild brain injury independently contribute to the risk of similar to those found in other developed countries andepilepsy.25 Thus, people genetically predisposed to indicate a high completeness.10 If cases with epilepsy areepilepsy (ie, with a family history of epilepsy) have a missed in the Danish National Hospital Register, thehigher risk of epilepsy than do people without genetic relative risk of epilepsy would be affected only if thepredisposition when exposed to mild brain injury. To incomplete capture of patients differs between thoseour knowledge, no previous studies have studied the with and without brain injury. Patients with head injuryrisk of epilepsy after brain injury in first degree relatives may be followed more closely than the generalto patients with epilepsy. In animals, variation in the population, which might increase the completeness andsusceptibility of various rat strains to post-traumatic overestimate the relative risk of epilepsy after brainepilepsy might lend some support to the hypothesis of an injury. However, the effect of this bias is likely tounderlying genetically determined tendency to develop decrease over time.post-traumatic epilepsy.26 Although, most patients with epilepsy are cared for on Our registration of family history is not complete an outpatient basis, the incidence estimate only increasedbecause some parents and older siblings might have by 17% after inclusion of outpatients.10 Hence, mostbeen diagnosed before the Danish National Hospital outpatients with epilepsy are also admitted to hospitalRegister was established (Jan 1, 1977). This mis- for that or other reasons and, thereby, included in theclassification is likely to cause an underestimation of the National Hospital Register. Some patients with severeeffect of family history on the risk of epilepsy. brain injury live in care homes in the community after The relative risk of epilepsy after mild brain injury was their condition has stabilised, but these patients have theslightly higher in female than in male patients perhaps same access to the hospital system as patients withoutbecause female patients with epilepsy are more likely brain injury, and thus we think that they do not have aregistered in the National Hospital Register because of decreased likelihood of being registered with epilepsy insex-specific factors, such as pregnancy. Alternatively the Danish National Hospital Register.female brains might be more susceptible to epilepsy after People were censored when they died or left Denmarkmild brain injury than are male brain, as supported by a permanently, but less than 3% of the entire cohort didprevious study showing that localisation-related epilepsy so.8 Some people may have had a brain injury or epilepsywith no apparent structural cause is more prevalent in during a short stay abroad; but numbers are likely to bewomen than in men.27 The sex difference was not present very low, and most of these will be treated in Danishfor the other types of brain injury, suggesting that other hospitals or outpatient clinics when they return tomechanisms might be involved in post-traumatic epilepsy Denmark. Bias due to selection of study participants isafter skull fracture and more severe brain injuries. therefore an unlikely explanation for our findings. In The length of first hospital stay with brain injury was comparison, 1139 (25%) patients of a total population ofassociated with an increased risk of epilepsy for severe 4541 in the Rochester study were lost to follow-up due tobrain injury and cranial fractures. The length of migration from Minnesota.1admission is probably related to severity of brain injury. A previous study assessed the validity of the hospital Despite the length and completeness of follow up, the codes for brain injury (ICD-8: 851–854) showing that thesize of the study cohort, and the population-based nature diagnoses were confirmed in about 88% of cases.29 However,of the study,8 we had limited clinical information. In a clinical discrimination between different types of brainrecent study, we validated the epilepsy diagnosis in the injury is difficult and the definitions vary betweenDanish National Hospital Register.11 We found a positive countries.14 Brain injuries that at first seem mild can turnpredictive value of an ICD-8 or ICD-10 epilepsy diagnosis out to be severe. In a study of 24 patients with post-traumaticaccording to ILAE criteria28 of 81% for epilepsy and amnesia lasting more than 1 week, four had initially been89% for single seizures, but identified no epilepsy diagnosed with skull fracture and four with concussion.14diagnoses based on acute symptomatic seizures.11 Thus, Although, there is debate about the importance ofsome patients registered with epilepsy in the present post-traumatic amnesia in the diagnosis of patients withstudy do not fulfil the diagnostic criteria, but the mild brain injury,14,30 some patients diagnosed with mildmisclassification would only bias the results of the head injury might actually suffer from more severe brainpresent study away from the null hypothesis if the injury, which would likely lead to an overestimated risk ofquality of the epilepsy registration differs between epilepsy associated with mild brain injury.www.thelancet.com Vol 373 March 28, 2009 1109
    • Articles In the study period (1977–2002), the incidence of mild 10 Christensen J, Vestergaard M, Pedersen MG, Pedersen CB, Olsen J, brain injury decreased,31 probably because fewer children Sidenius P. Incidence and prevalence of epilepsy in Denmark. Epilepsy Res 2007; 76: 60–65. were injured or because the need for observation decreased 11 Christensen J, Vestergaard M, Olsen J, Sidenius P. Validation of after introduction of new diagnostic methods, most notably epilepsy diagnoses in the Danish National Hospital Register. CT and MRI.14,32 If only the more severe mild head injuries Epilepsy Res 2007; 75: 162–70. 12 Vestergaard M, Pedersen CB, Sidenius P, Olsen J, Christensen J. were treated in hospitals in later year, it might explain the The long-term risk of epilepsy after febrile seizures in susceptible increased risk of epilepsy after brain injury in 1994–2002. subgroups. Am J Epidemiol 2007; 165: 911–18. However, the diagnostic criteria15,29 might have changed 13 Sun Y, Vestergaard M, Pedersen CB, Christensen J, Olsen J. Apgar scores and long-term risk of epilepsy. Epidemiology 2006; 17: 296–301. when the classification codes were changed from ICD-8 to 14 Engberg A. Severe traumatic brain injury—epidemiology, external ICD-10 in 1994,10,11 and completeness of epilepsy in the causes, prevention, and rehabilitation of mental and physical Danish National Hospital Register might have increased sequelae. Acta Neurol Scand 1995; 164 (suppl): 1–151. when outpatients were included in 1995.10 In the analyses, 15 Engberg A, Teasdale TW. Traumatic brain injury in children in Denmark: a national 15-year study. Eur J Epidemiol 1998; 14: 165–73. we tried to take these factors into account by adjusting for 16 American Congress of Rehabilitation Medicine. Definition of mild calendar year. traumatic brain injury. J Head Trauma Rehabil 1993; 8: 86–88. We know, that about 40–50% of all hospitalisations with 17 Pinner M, Børgesen SE, Jensen R, Birket-Smith M, Gade A, Riis JØ. traumatic brain injuries are related to road-traffic accidents, Konsensusrapport om commotio cerebri (hjernerystelse) og det postcommotionelle syndrom. http://www.vfhj.dk/admin/write/ 20–25% to falls, 8–10% to firearms and assaults, and the files/456.pdf (accessed Jan 8, 2008). remaining related to other causes, such as sporting injuries 18 Breslow NE, Day NE. Statistical methods in cancer research: depending on age and social background.14,33 In this study, volume II—the design and analysis of cohort studies. IARC Sci Publ 1987; 82: 1–406. we did not have information on cause of brain injury, but 19 Clayton D, Hills M. Statistical models in epidemiology. Oxford: prevention measures such as the use of bicycle helmets34,35 Oxford University Press, 1993. might prevent brain injury and subsequent epilepsy, 20 Breslow NE. Generalized linear models: checking assumptions and strengthening conclusions. Statistica Applicata 1996; 8: 23–41. although the effectiveness of such measures has been 21 Pitkanen A, McIntosh TK. Animal models of post-traumatic questioned.36,37 epilepsy. J Neurotrauma 2006; 23: 241–61. Traumatic brain injury is a significant risk indicator for 22 Benardo LS. Prevention of epilepsy after head trauma: do we need epilepsy many years after the injury. Drug treatment after new drugs or a new approach? Epilepsia 2003; 44 (suppl 10): 27–33. brain injury with the aim of preventing post-traumatic 23 Behandling af traumatiske hjerneskader og tilgrænsende lidelser. The Danish Board of National Health. 1-208. The Danish National epilepsy has been discouraging, but our data suggest a Board of Health, 1997. long time interval for potential, preventive treatment of 24 Kjeldsen MJ, Corey LA, Christensen K, Friis ML. Epileptic seizures high risk patients. and syndromes in twins: the importance of genetic factors. Epilepsy Res 2003; 55: 137–46. Contributors 25 Rothman KJ. Modern epidemiology. Boston: Little Brown, 1986. JC and MV initiated the study and obtained funding. MV, CBP, MGP, JO, 26 Berkovic SF, Mulley JC, Scheffer IE, Petrou S. Human epilepsies: PSI, and JC designed the study. MGP and CBP constructed the population. interaction of genetic and acquired factors. Trends Neurosci 2006; JC, MGP, CBP, and MV analysed the data. JC, MV, and CBP wrote the first 29: 391–97. draft; JC wrote the revised versions. All authors interpreted the results, 27 Christensen J, Kjeldsen MJ, Andersen H, Friis ML, Sidenius P. revised the paper, and approved the final version. Gender differences in epilepsy. Epilepsia 2005; 46: 956–60. Conflict of interest statement 28 Commission on Classification and Terminology of the International League Against Epilepsy. Proposal for revised classification of We declare that we have no conflict of interest. epilepsies and epileptic syndromes. Epilepsia 1989; 30: 389–99. References 29 Engberg AW, Teasdale TW. Traumatic brain injury in Denmark 1 Annegers JF, Hauser WA, Coan SP, Rocca WA. A population-based 1979–1996: a national study of incidence and mortality. study of seizures after traumatic brain injuries. N Engl J Med 1998; Eur J Epidemiol 2001; 17: 437–42. 338: 20–24. 30 Bruns TJ, Hauser WA. The epidemiology of traumatic brain injury: 2 Frey LC. Epidemiology of posttraumatic epilepsy: a critical review. a review. Epilepsia 2003; 44: 2–10. Epilepsia 2003; 44 (suppl 10): 11–17. 31 Engberg AW, Teasdale TW. Epidemiology and treatment of head 3 Angeleri F, Majkowski J, Cacchio G, et al. Posttraumatic epilepsy risk injuries in Denmark 1994–2002, illustrated with hospital statistics. factors: one-year prospective study after head injury. Epilepsia 1999; Ugeskr Laeger 2007; 169: 199–203. 40: 1222–30. 32 Metting Z, Rodiger LA, De Keyser J, van der Naalt J. Structural and 4 Englander J, Bushnik T, Duong TT, et al. Analyzing risk factors for functional neuroimaging in mild-to-moderate head injury. late posttraumatic seizures: a prospective, multicenter investigation. Lancet Neurol 2007; 6: 699–710. Arch Phys Med Rehabil 2003; 84: 365–73. 33 Thurman DJ, Alverson C, Dunn KA, Guerrero J, Sniezek JE. 5 D’Ambrosio R, Perucca E. Epilepsy after head injury. Curr Opin Neurol Traumatic brain injury in the United States: a public health 2004; 17: 731–35. perspective. J Head Trauma Rehabil 1999; 14: 602–15. 6 Agrawal A, Timothy J, Pandit L, Manju M. Post-traumatic epilepsy: an 34 Macpherson A, Spinks A. Bicycle helmet legislation for the uptake overview. Clin Neurol Neurosurg 2006; 108: 433–39. of helmet use and prevention of head injuries. 7 Temkin NR. Antiepileptogenesis and seizure prevention trials with Cochrane Database Syst Rev 2008; 3: CD005401. antiepileptic drugs: meta-analysis of controlled trials. Epilepsia 2001; 35 Thompson DC, Rivara FP, Thompson R. Helmets for preventing 42: 515–24. head and facial injuries in bicyclists. Cochrane Database Syst Rev 8 Pedersen CB, Gotzsche H, Moller JO, Mortensen PB. The Danish 2000; 2: CD001855. Civil Registration System: a cohort of eight million persons. 36 Hewson PJ. Cycle helmets and road casualties in the UK. Dan Med Bull 2006; 53: 441–49. Traffic Inj Prev 2005; 6: 127–34. 9 Andersen TF, Madsen M, Jorgensen J, Mellemkjoer L, Olsen JH. 37 Robinson DL. No clear evidence from countries that have enforced The Danish National Hospital Register: a valuable source of data for the wearing of helmets. BMJ 2006; 332: 722–25. modern health sciences. Dan Med Bull 1999; 46: 263–68.1110 www.thelancet.com Vol 373 March 28, 2009
    • Comment be able to provide a definitive treatment decision.9 To 3 Verweij J, Casali PG, Zalcberg J, et al. Progression-free survival in gastrointestinal stromal tumours with high-dose imatinib: randomised refine the indication for adjuvant treatment remains the trial. Lancet 2004; 364: 1127–34. big task for futures studies. 4 Debiec-Rychter M, Sciot R, Le Cesne A, et al, on behalf of the EORTC Soft Tissue and Bone Sarcoma Group, The Italian Sarcoma Group and the Australasian GastroIntestinal Trials Group. KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal Peter Hohenberger tumours. Eur J Cancer 2006; 42: 1093–103. Division of Surgical Oncology and Thoracic Surgery, 5 Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and Department of Surgery, Medical Faculty Mannheim, prognosis at different sites. Semin Diagn Pathol 2006; 23: 70–83. University of Heidelberg, D-68135 Mannheim, Germany 6 Corless CL, Schroeder A, Griffith D, et al. PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro peter.hohenberger@chir.ma.uni-heidelberg.de sensitivity to imatinib. J Clin Oncol 2005; 23: 5357–64. I have received research grants and honoraria from Novartis. 7 Mussi C, Schildhaus HU, Gronchi A, Wardelmann E, Hohenberger P. Therapeutic consequences from molecular biology for GIST patients 1 Casali PG, Jost L, Reichardt P, Schlemmer M, Blay J-Y, on behalf of the ESMO affected by neurofibromatosis type 1. Clin Cancer Res 2008; 14: 4550–55. Guidelines Working Group. Gastrointestinal stromal tumors: ESMO clinical 8 Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal recommendations for diagnosis, treatment and follow-up. Ann Oncol stromal tumors: a consensus approach. Hum Pathol 2002; 33: 459–65. 2008; 19 (suppl 2): ii35–38. 9 Gronchi A, Judson I, Nishida T, et al. Adjuvant treatment of GIST with 2 DeMatteo RP, Ballman KV, Antonescu CR, on behalf of the American imatinib: solid ground or still quicksand? A comment on behalf of the EORTC College of Surgeons Oncology Group (ACOSOG) Intergroup Adjuvant GIST Soft Tissue and Bone Sarcoma Group, the Italian Sarcoma Group, the NCRI Study Team. Adjuvant imatinib mesylate after resection of localised, Sarcoma Clinical Studies Group (UK), the Japanese Study Group on GIST, the primary gastrointestinal stromal tumour: a randomised, double-blind, French Sarcoma Group and the Spanish Sarcoma Group (GEIS). Eur J Cancer placebo-controlled trial. Lancet 2009; published online March 19. 2009; published online March 16. DOI:10.1016/j.ejca.2009.02.009. DOI:10.1016/S0140-6736(09)60500-6. Risk of epilepsy after head trauma Published Online Head trauma is an important cause of epilepsy, and for 2–3 years after a severe head injury, but the excess February 23, 2009 DOI:10.1016/S0140- knowledge of the extent of the risk of epilepsy after risk continued for 10 years after mild and severe brain 6736(09)60215-4 head trauma and the factors that influence this risk injury—longer than in other studies.2 The incidence See Articles page 1105 are essential. In The Lancet today, Jakob Christensen of epilepsy was greater in head-injured people with a and colleagues1 present their population-based cohort family history of epilepsy than in those without a family study of more than 1·5 million people born in Denmark history, with about a six-fold increase in the relative between 1977 and 2002, and followed up for that risk of epilepsy after a mild head injury and a ten-fold period. 78 572 of them had at least one head injury increase after a severe injury. This finding emphasises and 17 470 were diagnosed with epilepsy, of whom that the cause of epilepsy is often multifactorial. 1017 had had a head injury before diagnosis. These Previous studies in this area have been either too researchers obtained the data from the Danish National small or open to too many methodological criticisms to Hospital Register, which provided diagnostic coding on be deemed to provide definitive data. Christensen and inpatients from 1977, and outpatients from 1995, on co-workers’ investigation is of commendable size and epilepsy and head injury. Family history was ascertained completeness, with an advanced statistical design—as by linkage of data from first-degree relatives. The such, it should be accepted as the reference study in the researchers compared the relative risks of development field. This is not to say that there are not methodological of epilepsy for people with mild and severe head injury criticisms. There are issues inherent in the study design: (with or without a family history of epilepsy) on a yearly the diagnosis of epilepsy and the classification of basis with those for people without head injury, while severity of trauma are based on registry data, with all controlling for age, sex, and calendar year. the inaccuracy that this implies; no attempt is made Overall, the relative risks of epilepsy were raised about to distinguish between immediate, early, and late two-fold (relative risk 2·2) after a mild head injury and epilepsy although these categories have important seven-fold (7·4) after a severe head injury, were slightly clinical implications; previously identified risk factors greater in women than in men, and increased with for post-traumatic epilepsy, such as the presence of older age at time of injury. The rate of development of dural tear, intracranial haemorrhage, and early seizures epilepsy was greatest in the few years after the head (<1 week) were not investigated; and no data are injury; for instance, with a greater than five-fold increase provided about the type or severity of the epilepsy.1060 www.thelancet.com Vol 373 March 28, 2009
    • Comment the possibility that neuroprotective measures3 could interfere with this process and thus reduce the risk of epilepsy. Past attempts to prevent epilepsy have been disappointing,4 but these new data suggest that such efforts should be renewed, to focus particularly on high-risk groups (those with severe head injury, within 2 years of injury, and a positive family history). Finally, we should note the value of such large-scale epidemiological studies that use pre-existing databases. Such studies are increasingly difficult to do in the UK, for example, because of sometimes over-zealous inter- pretation of confidentiality and consent regulations, and the timidity of the bureaucratic processes. In the UK, we have reached a situation in which, in large swathes of clinical epidemiological research, the baby is being well and truly thrown out with the bathwater, to the detriment of patients and the acquisition ofScience Photo Library beneficial knowledge.5 *Simon Shorvon, Aidan Neligan Subdural haematoma (red) in 10-year-old boy following trauma University College London Institute of Neurology, National Hospital for Neurology and Neurosurgery, London WC1N 3BG, UK s.shorvon@ion.ucl.ac.uk The decision about whether or not to give anti- We declare that we have no conflict of interest. epileptic drugs prophylactically in patients with head 1 Christensen JC, Pedersen MG, Pedersen CB, Sidenius P, Olsen J, injury is a common clinical dilemma. Christensen Vestergaard M. Long-term risk of epilepsy after traumatic brain injury in children and young adults: a population-based cohort study. Lancet and co-workers’ study does not address the value of 2009; published online Feb 23. DOI:10.1016/S0140-6736(09)60214-2. treatment, but the risk estimates will help patients 2 Annegers JF, Hauser WA, Coan SP, Rocca WA. A population-based study of seizures after traumatic brain injuries. N Engl J Med 1998; and doctors make decisions more clearly. The study will 338: 20–24. also be of value in helping to determine epilepsy risks 3 Temkin NR. Antiepileptogenesis and seizure prevention trials with antiepileptic drugs: meta-analysis of controlled trials. Epilepsia 2001; for medicolegal purposes, by providing a sound basis 42: 515–24. for determination of cause and compensation and, as 4 Temkin NR, Dikmen SS, Wilensky AJ, Keihm J, Chabal S, Winn HR. A randomized, double-blind study of phenytoin for the prevention of such, is a service to social justice. Scientific value exists post-traumatic seizures. N Engl J Med 1990; 323: 497–502. 5 Metcalfe C, Martin RM, Noble S, et al. Low risk research using routinely too in the finding that the risk of epilepsy was increased collected identifiable health information without informed consent: for at least 10 years after head injury. Post-traumatic encounters with the Patient Information Advisory Group. J Med Ethics 2008; 34: 37–40. epileptogenesis is thus a long process, which raises Elimination of blinding trachoma revolves around children Blinding trachoma is a terrible disease. The intense older people have trichiasis. Trachoma is now restricted See Articles page 1111 conjunctival inflammation in young children causes to poor developing areas, having disappeared from conjunctival scarring, leading in adult life to inturned Europe and North America where only a century ago it eyelashes (trichiasis) that rub on the eye and cause was a major problem. painful blindness. In The Lancet today, Jenafir House and Chlamydia trachomatis, the causative bacterium colleagues report a study in hyperendemic communities for trachoma, has evolved with human beings and in Ethiopia.1 In such areas more than half of children are their vertebrate ancestors since Jurassic times.2 It has affected, almost every adult has scarring, and 10–20% of developed an effective host–parasite relation over a www.thelancet.com Vol 373 March 28, 2009 1061
    • BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –1 82 a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e sResearch ReportNeuroprotective effect of memantine combined withtopiramate in hypoxic–ischemic brain injuryChunhua Liu, Niyang Lin⁎, Beiyan Wu, Ye QiuDepartment of Pediatrics, The First Affiliated Hospital of Shantou University Medical College, 515000, Shantou, ChinaA R T I C LE I N FO AB S T R A C TArticle history: Glutamate receptor-mediated neurotoxicity is a major mechanism contributing to hypoxic–Accepted 20 May 2009 ischemic brain injury (HIBI). Memantine is a safe non-competitive NMDA receptor blockerAvailable online 6 June 2009 characterized by its low affinity and fast unblocking kinetics. Topiramate is an AMPA/KA receptor blocker and use-dependent sodium channel blocker with several other neuropro-Keywords: tective actions and little neurotoxicity. We hypothesized that the coadministration ofNeuroprotection memantine and topiramate would be highly effective to attenuate HIBI in neonatal rats.Memantine Seven-day-old Sprague–Dawley rat pups were subjected to right common carotid arteryTopiramate ligation and hypoxia for 2 h, and then were randomly and blindly assigned to one of fourPharmacology groups: vehicle, memantine, topiramate and combination group. Brain injury was evaluatedCombination therapy by gross damage and weight deficit of the right hemisphere at 22d after hypoxic-ischemiaGlutamate receptor (HI) and by neurofunctional assessment (foot-fault test) at 21d post-HI. Acute neuronal injuryApoptosis was also evaluated by microscopic damage grading at 72 h post-HI. Results showed the combination of memantine and topiramate improved both pathological outcome and performance significantly. The drug-induced apoptotic neurodegeneration was assessed by TUNEL staining at 48 h post-HI and the result showed no elevated apoptosis in all observed areas. The result of the experiment indicates the combination therapy is safe and highly effective to reduce brain damage after HIBI. © 2009 Elsevier B.V. All rights reserved.1. Introduction voltage-sensitive Ca2+ channels, and Ca2+-permeable AMPA/ KA channels (Lu et al., 1996). The excitotoxic overactivation ofThe excessive glutamate release and overactivation of gluta- NMDA and AMPA/KA glutamate receptors provokes furthermate receptors are crucial contributors to the pathogenesis of glutamate release and further NMDA and AMPA/KA receptorHIBI. They cause a massive influx of sodium (Na+) and calcium stimulation, and it forms a positive feedback cycle making the(Ca2+) that triggers a cascade of biochemical events, and lead condition worse (Villmann and Becker, 2007). To break thisto neuronal necrosis and apoptosis in many types of cells in vicious cycle, researchers utilized many NMDA and/or AMPA/KAneonatal brain. There are three subtypes of ionotropic receptor antagonists, but most of them have severe side effectsglutamate receptors involved, namely N-methyl-D-aspartate (Haberny et al., 2002; Puka-Sundvall et al., 2000). For example, a(NMDA) receptors, α-3-amino-hydroxy-5-methyl-4-isoxazole widely used NMDA antagonist MK-801 was found inducingpro-pionic acid (AMPA) receptors and kainate (KA) receptors. widespread apoptotic neurodegeneration and impairing manyExcitotoxic injury occurs secondary to glutamate-triggered normal neuronal functions in developing rat brain (IkonomidouCa2+ influx through any of three routes: NMDA channels, et al., 1999). As NMDA receptors are essential for normal ⁎ Corresponding author. Fax: +86 754 88980347. E-mail address: niyanglin@hotmail.com (N. Lin).0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.brainres.2009.05.071
    • 174 BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –18 2 Table 1 – Neurologic damage score. Group No. Normal = 1 Mild = 2 Moderate = 3 Severe = 4 p⁎ Vehicle 19 4 4 3 8 NS Memantine 24 10 7 4 3 <0.05 Topiramate 21 5 5 5 6 >0.05 Combination 24 13 6 4 1 <0.01 The number of pups receiving the designated gross damage score by a blinded observer. ⁎ p value, memantine, topiramate, combination vs. vehicle.physiological processes in brain development, including the associated with an increase in the intensity and number ofproliferation, migration, survival and differentiation of neurons, synaptic AMPA-receptor clusters (Liao et al., 2001; Liu et al.,blockade of excessive NMDA receptor activity must be achieved 2004a). These findings suggest that it will be more effectivewithout affecting normal brain functioning (Kohr, 2007). and beneficial to block both NMDA and AMPA/KA receptors by Recently, increasing evidence based on molecular studies combination of different glutamate receptor antagonists.suggests that memantine, an uncompetitive NMDA receptor Based on the pharmacology and mechanism studies, weblocker with fast channel unblocking kinetics to prevent it from designed the experiments to evaluate the efficacy of theoccupying the channels and interfering with normal synaptic combination therapy by measuring gross brain damage, braintransmission, is a potent neuroprotectant without above- weight deficit in the right hemisphere and regional neuronalmentioned side effects (Chen et al., 1992, 1998; Chen and Lipton, injury. Besides the morphologic and histopathologic measure-2005; Johnson and Kotermanski, 2006). In contrast to MK-801 ment, a neurofunctional test was performed to verify theand ketamine, memantine shows unusual clinical tolerance in results. To ensure therapeutic safety, the possible drug-the treatment of moderate-to-severe Alzheimers disease in induced apoptosis was assessed even though the two drugsadults through its low affinity and relatively fast unblocking were approved safe and efficient in their respective therapeu-kinetics (de Lima et al., 2000; Lipton, 2004; Lipton, 2006). As a tic categories (Chen et al., 1998; Glier et al., 2004).neuroprotective agent, memantine can reduce functional aswell as morphological sequelae induced by ischemia (Block andSchwarz, 1996; Chen et al., 1998). A recent study showed the 2. ResultsNMDA receptor blockade with memantine could provide aneffective pharmacological prevention of periventricular leuko- 2.1. Gross brain damage gradingmalacia (PVL) in the premature infant (Manning et al., 2008). Topiramate, a well tolerated antiepileptic drug (AED) used The neurologic damage score was determined by an observerclinically, confers neuroprotection by blocking AMPA/KA blind to the drug treatment of the rat pups. Table 1 shows thereceptors and use-dependent Na+ channel in developing rat neurologic damage scores in each group. The neurologicbrain without serious side effects compared to conventional damage score was significantly higher in the vehicle-treatedanticonvulsants (Noh et al., 2006). Topiramate has anti- group (2.79 ± 1.23, n = 19) than that in the combination-treatedexcitotoxic properties, because it protects against motorneuron degeneration. The other neuroprotective effects oftopiramate include positive modulation of gamma-aminobu-tyric acid (GABA) receptors, increase of seizure threshold andso on (Pappalardo et al., 2004). Furthermore, Topiramate alsoprotects preoligodendrocytes against excitotoxic cellulardeath in white matter lesions and prevents the periventricularwhite matter from the damage induced by an AMPA/KAagonist in newborn mice (Follett et al., 2004; Sfaello et al., 2005). Due to the complex pathological mechanisms in HIBIdescribed above, combination therapy or multimodal target-ing is thought to be a key future approach to provide effectiveneuroprotection. Most promising combination should targetdifferent neuroprotective mechanisms, expand the therapeu-tic time window, and alleviate the possibility of side effects Fig. 1 – The percentage of reduction in right cerebral(Rogalewski et al., 2006). Studies on the mechanisms of the hemisphere weight measured using the left hemispheresuperfamily of glutamate receptors revealed that NMDA and weight as standard. The animal numbers are as described inAMPA glutamate receptors showed a fine-tuned interaction at the result. The percentage of reduction in right hemispherethe glutamatergic synapse: the rapid activation and brief open weight was significantly decreased in the combination grouptime of AMPA receptors facilitates unblock of NMDA receptors compared with the vehicle group (**p < 0.01 vs. vehicle). The(Villmann and Becker, 2007). Functional interdependence of percentage of reduction in right hemisphere weight wasAMPA and NMDA receptors has been proven by experiments significantly decreased in the memantine group comparedwhere a transient synaptic activation of NMDA receptors with the vehicle group (*p < 0.05 vs. vehicle). Data arereliably induces a long-term potentiation phenomenon, presented as mean ± S.D.
    • BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –1 82 175Fig. 2 – Microscopic brain damage scores in the cortex, hippocampus, striatum, thalamus. Data are presented as mean ± S.D.*p < 0.05 vs. vehicle, **p < 0.01 vs. vehicle.group (1.71 ± 0.91, n = 24, p < 0.01 versus vehicle). The neurologic group (2.15± 0.52 and 1.51± 0.47, n = 12, p < 0.05 and p < 0.05 versusdamage score was significantly higher in the vehicle-treated vehicle) was significantly lower compared with the vehicle-group than that in the memantine-treated group (2.00 ± 1.06, treated group (4.15± 0.73 and 3.38± 0.72, n = 10) in the cortex andn = 24, p < 0.05 versus vehicle). The neurologic damage score in thalamus. The histopathologic score in the combination-treatedthe topiramate-treated group (2.57 ± 1.17, n = 21, p > 0.05 versus group (1.91 ± 0.51, 1.45 ± 0.49 and 0.91 ± 0.42, n = 12, p < 0.05,vehicle) was lower but not statistically significant compared p < 0.05 and p < 0.01 versus vehicle) was significantly lowerwith the vehicle-treated group. compared with the vehicle-treated group (4.15± 0.73, 3.68 ± 0.62 and 3.38 ± 0.72, n = 10) in the cortex, hippocampus and thalamus.2.2. Brain weight deficit In the striatum, the histopathologic score in the combination- treated group was lower but not statistically significantFig. 1 shows the weight deficit in the right hemisphere relative compared with the vehicle-treated group.to the left hemisphere. The weight deficit in the combination-treated group (9.2 ± 2.5%, n = 24, p < 0.01 versus vehicle) was 2.4. Foot-fault testsignificantly reduced compared with the vehicle-treatedgroup (26.9 ± 4.1%, n = 19). The weight deficit in the meman- Fig. 3 shows the number of foot-faults in each group. Thetine-treated group (16.3 ± 3.2%, n = 24, p < 0.05 versus vehicle) number of foot-faults per pup was significantly greater in thewas significantly reduced compared with the vehicle-treated vehicle-treated group (8.62 ± 1.51, n = 10) than that in thegroup. The weight deficit in the topiramate-treated group(21.5 ± 4.0%, n = 21, p > 0.05 versus vehicle) was reduced butnot statistically significant compared with the vehicle-treated group. Body weights of rat pups in each group wererecorded and analyzed. Results showed that the bodyweights of the treated groups were not significantly differentfrom the vehicle-treated group at 1, 3, 7, 14 and 22 days afterinjury (data not shown). Mortality rates were not signifi-cantly different in four groups, although there was a trendtoward reduced mortality in the combination group.2.3. Microscopic brain damage gradingThe microscopic brain damage score (histopathologic score) wasdetermined by an observer blind to the drug treatment of the rat Fig. 3 – Number of foot-faults in each group. The combinationpups. Fig. 2 shows the microscopic brain damage score in each group had significantly fewer foot-faults than the vehiclegroup. The histopathologic score in the memantine-treated group. Data are presented as mean ± S.D. *p < 0.05 vs. vehicle.
    • 176 BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –18 2Fig. 4 – The numbers of TUNEL-positive apoptotic cells in the cortex, the CA1, CA3 and dentate gyrus of the hippocampus, thestriatum and the subcortical white matter in the vehicle, memantine, topiramate and combination group. Data are presented asmean ± S.D. *p < 0.05 vs. vehicle, **p < 0.01 vs. vehicle.combination-treated group (4.26 ± 0.93, n = 12, p < 0.05 versus apoptosis are shown in Fig. 5. In all observed areas, thevehicle). The number of foot-faults per pup was significantly numbers of apoptotic cells in the treated group (single orgreater in the vehicle-treated group than that in the meman- combined) were not significantly increased compared with thetine-treated group (4.66 ± 1.03, n = 12, p < 0.05 versus vehicle). vehicle-treated group. In the CA1 sector of the hippocampus,The number of foot-faults per pup was less but not statistically The numbers of apoptotic cells in the combination-treatedsignificant in the topiramate-treated group (6.94 ± 1.22, n = 11) group (31.2 ± 20.7 and 45.5 ± 31.2, n = 12, p < 0.01 and p < 0.01compared with in the vehicle-treated group. versus vehicle) were significantly reduced compared with the vehicle-treated group (82.1 ± 32.6 and 175 ± 48.2, n = 12). In the2.5. TUNEL-positive cell counting CA1 sector of the hippocampus and the subcortical white matter, The numbers of apoptotic cells in the memantine-The numbers of TUNEL-positive apoptotic cells of each group treated group (50.5 ± 28.3 and 99.8 ± 38.7, n = 12, p < 0.05 andare presented in Fig. 4 and areas examined for drug-induced p < 0.05 versus vehicle) were significantly reduced compared with the vehicle-treated group. In other areas, no significant differences were found between any of the treated groups (single or combined) and the vehicle group. Fig. 6 shows some sample pictures of apoptotic cells in the CA1 sector of the hippocampus. 3. Discussion The present study shows for the first time to our knowledge that the combination of memantine and topiramate exerts enhanced protection of neurons against HIBI in vivo, compared with each of these agents alone. In this study, we measured brain damage in each group by using the gross anatomic method of Palmer et al. at 22d post-HI. By delaying assessment until 22d after HI, we included very late cell death that reflects overall neuroprotective effect of the drugs in a relatively long period. We also examined the brain weight deficit presentedFig. 5 – Areas of the brain examined for neuronal injury and by the loss of brain weight on the ipsilateral side relative to thedrug-induced apoptosis. CX = cortex CA1 = hippocampus CA1 contralateral side. Results showed the combination therapyCA3 = hippocampus CA3 Den = dentate gyrus ST = striatum significantly reduced the degree of brain injury in this model.TH = thalamus. Besides the morphologic examinations, we applied the foot-
    • BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –1 82 177Fig. 6 – Sample pictures of TUNEL-positive apoptotic cells in the CA1 sector of the hippocampus in the (A) vehicle,(B) memantine, (C) topiramate and (D) combination group. Original magnification, ×400.fault test to evaluate sensorimotor function of the rat pups at post-HI. Because short-term neuronal injury in the developing21d post-HI. Foot-faults per pup in the combination group brain after HI is caused by both early and delayed neurode-were significantly less than that in the vehicle group. The generation, the onset of damage in different regions of thefunctional outcome was consistent with the morphologic brain is time-dependent and progressive, and it has an unevenfindings in the long-term perspective. The short-term effect of distribution within regions (Northington et al., 2001). However,the combination therapy was evaluated by microscopic brain 72h (3d) post-HI seems an appropriate time point to evaluatedamage scoring at 72 h post-HI. Results showed that the short-term neuronal injury after insult in this model (Feng etcombination therapy reduced neuronal injury significantly in al., 2005, 2008; Manning et al., 2008; Zhu et al., 2004).the cortex, hippocampus and thalamus. In our experiment, the time window and doses of Neuronal cell death after HI has generally been attributed memantine and topiramate were chosen according to ato either rapid necrosis or delayed apoptosis. There is no doubt general purpose to achieve an application for potential clinicalthat necrosis plays major role in the course. But the develop- use. Based on published data of rat pharmacokinetics anding brain may have good plasticity and a high capacity for self- dose–response studies, 20 mg/kg dose of memantine canrepair (Daval et al., 2004; Grafe, 1994). After most compensa- provide minimal neuroprotection (Chen et al., 1998; Hesselinktory and reparative phrases have passed, there are at least et al., 1999). Considering the short therapeutic time windowthree different end points should be taken into account in (Culmsee et al., 2004) and the confirmed neuroprotectiveassessment: the long-term deficit of brain tissue, the func- effects of memantine at 20 mg/kg dose in HI and PVL model,tional consequences of the brain injury and the acute extent of we administered the 20 mg/kg loading dose of memantinebrain injury (Bona et al., 1997). The quantitive assessment of immediately after HI in the treatment. Topiramate (loadingbrain weight deficit and gross brain damage used in this study dose 50 mg/kg, maintenance dose 20 mg/kg/day) can reducecan accurately evaluate neuroprotective effects of glutamate neuronal cell loss significantly but increase apoptosis in theantagonists against NMDA-mediated brain injury in vivo frontal white matter in newborn piglets (Schubert et al., 2005).(Andine et al., 1990; McDonald et al., 1989a). On the other Furthermore, topiramate may cause neurodegeneration in thehand, behavioral consequences after HIBI are essential to developing rat brain only at doses at and above 50 mg/kgreveal the true functional disability and to study the effects of (Glier et al., 2004). The reason why topiramate at doses abovedrug intervention. In this study, the foot-fault test was done at 50 mg/kg can protect neurons but increase apoptosis may21d post-HI to evaluate the long-term functional outcome. relate to two mechanisms. The first one is the blockade ofDifferent from other cognitive function tests (Morris water AMPA/KA receptors lack of interference with NMDA-receptormaze, etc) related mostly to the hippocampus formation, the signaling (Gibbs et al., 2000). Topiramate cannot providefoot-fault test correlates with brain lesion in the cerebral neuroprotection only through AMPA/KA receptor channelcortex which is the most constantly affected region in both unless it reaches threshold dosage. The second one is themild and severe HIBI in this model (Bona et al., 1997). Short- depression of the endogenous neurotrophin system in theterm effect of the therapy was evaluated by a scoring system brain which may account for the proapoptotic effect (Bittigauon neuronal injury in 4 main regions of the rat brain at 72 h et al., 2002). In a gerbil model, topiramate was found reducing
    • 178 BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –18 2hippocampal neuronal damage in dose-dependent manner sible for the inactivation of glutamate as a neurotransmitter(Lee et al., 2000). Based on the dose–response studies and our (Poulsen et al., 2006). Moreover, topiramate was foundpreliminary experiment, we chose 40 mg/kg as the loading effective in attenuating seizure-induced neuronal cell deathdose for topiramate. The dose of topiramate (loading dose and reducing KA-induced Phospho-extracellular signal-regu-40 mg/kg; maintenance dose 20 mg/kg/day) was proven lated kinase-immunoreactive (p-Erk IR) in the CA3 region ofconsiderably safe but unlikely to be neuroprotective. the hippocampus (Park et al., 2008). In a rat pup model of PVL, Although the mechanisms underlying the neuroprotection topiramate has been demonstrated effective to attenuateare not fully understood, the results demonstrate that a AMPA/KA receptor-mediated cell death and Ca2+ influx, assynergistic reduction in brain damage can be achieved well as KA-evoked currents in developing oligodendrocyteseffectively by memantine combined with topiramate. The (Follett et al., 2004).neuroprotective actions and unique characteristics of these Many studies suggest that combination of drugs maytwo drugs may account for the experimental outcome. It is produce greater toxicity than individual ones. Thus, the safetywell documented that memantine antagonizes NMDA recep- of combination therapies should be most concerned, whentor activation by inhibiting the influx of Ca2+ through this these animal findings are intended for extrapolating to achannel (Johnson and Kotermanski, 2006). As an open- pediatric surgical patient population (Bittigau et al., 2002). Thechannel blocker, memantine can provide neuroprotection rat is most sensitive to NMDA receptor-mediated neurotoxi-without interference with the normal brain development city during early neuronal pathway development, referred to(Parsons et al., 1999). The favorable kinetics of memantine as the “brain-growth spurt period” or period of synaptogen-interaction with NMDA channels may be partly responsible for esis. (Haberny et al., 2002). Blockade of NMDA receptors up toits high index of therapeutic safety, and it makes memantine a 4 h is sufficient to trigger apoptotic neurodegeneration in thecandidate drug for use in many NMDA receptor-mediated developing brain (Ikonomidou et al., 1999). In consideration ofhuman CNS disorders (Johnson and Kotermanski, 2006; the possible neurotoxicity caused by the coadminstration ofLipton, 2004). In a four-vessel-occlusion (4VO) global ischemic drugs and the complicated interaction between NMDA recep-model, neuronal damage in the CA1 sector of the hippocam- tor blocker and AMPA receptor blocker, we examined thepus and in the striatum produced by 4VO was significantly possible drug-induced neuronal apoptosis by TUNEL stainingattenuated by 20 mg/kg memantine (Block and Schwarz, 1996). at 48 h post-HI even through the two drugs are proven safe atMemantine has been used clinically for excitotoxic disorders the given doses respectively (Chen et al., 1998; Glier et al.,at neuroprotective doses administered up to 2 h after 2004). The time course of apoptotic injury varies regionallyinduction of HI in immature and adult rats. At neuroprotective because HI damage generally evolves more rapidly in theconcentrations, memantine results in few adverse side effects immature brain than its adult counterpart. Injury in the cortexand displays virtually no effects on Morris water maze and striatum occurs in a biphasic manner, where the earlyperformance or on neuronal vacuolation (Chen et al., 1998). phase (by 3 h) is classified as necrosis and the later phase (byRosi et al. found that memantine protects against LPS-induced 48 h) displays signs of apoptosis (Northington et al., 2001).neuroinflammation, and confers neural and cognitive protec- Nakajima et al. found that the density of caspase-3 immunor-tion (Rosi et al., 2006). Furthermore, NMDA receptor blockade eactivity was enhanced in the frontal, parietal, and cingulatewith memantine can provide an effective pharmacological cortex and in the striatum 24 h after hypoxic ischemic injury.prevention of PVL in the premature infant without affecting In the CA3 sector of the hippocampus, the dentate gyrus,normal myelination or cortical growth (Manning et al., 2008). medial habenula and laterodorsal thalamus, the density of Topiramate is a novel broad spectrum antiepileptic drug apoptotic cells was highest at 24–72 h after HI and then(AED) used clinically in adults and children older than 2 years. declined. In thalamus, increased caspase-3 immunoreactivityAmongst new-generation AEDs examined for neurotoxicity in was distributed in lateral, laterodorsal, and reticular nucleineonatal rats, topiramate holds promise for minimizing the with a peak in density at 48 h after HI. In hippocampus,risk of neuronal death without side effects such as the intense caspase-3 immunoreactivity was present in CA1 andimpairment of cognitive performance (Cha et al., 2002; Glier in the dentate gyrus at 48 h after insult but had nearlyet al., 2004; Mellon et al., 2007). Pharmacological actions of disappeared by 7d after HI injury (Nakajima et al., 2000).topiramate include positive modulation of GABA receptors, Based on all these results on apoptotic injury, the time pointinhibition of the AMPA/KA glutamate receptor subtypes and (48 h post-HI) was chosen to examine the apoptoticblockade of a use-dependent Na+ channel (Schubert et al., neurodegeneration.2005). Noh and his coworkers reported the co-treatment of In this experiment, massive cellular apoptosis was nottopiramate and an NMDA receptor antagonist D-AP5 greatly found in all observed areas in the treated groups, andincreased the number of viable neurons in oxygen–glucose apoptosis was reduced in the CA1 sector of the hippocampusdeprivated cells. The experiment determined that neuropro- and the subcortical white matter in the combination grouptective effect of topiramate was mainly mediated by the compared with the vehicle group. The safe dosing regimeninhibition of AMPA glutamate receptors (Noh et al., 2006). and anti-apoptotic actions of memantine and topiramate mayTopiramate blocks the spread of seizures caused by transient contribute to the results synergistically. Regional patterns ofglobal cerebral ischemia, and reduces the abnormally high neuronal death can also be detected by expression of caspase-extracellular levels of glutamate in the hippocampus in the 3, a cysteine protease involved in the execution phase ofimmature rat spontaneous epileptic model by blocking AMPA apoptosis. Immunocytochemical and Western blot analysesreceptors (Koh et al., 2004). It also affects the expression of show increased caspase-3 expression in damaged hemi-glutamate transporters (GLAST and GLT-1) which are respon- spheres 24 h to 7d after HI. Reduced caspase-3 activity has
    • BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –1 82 179been shown to be associated with neuroprotection (Endres et assigned to one of the following groups: vehicle group (saline),al., 1998; Puka-Sundvall et al., 2000). Memantine (20 mg/kg, i. memantine group, topiramate group, combination groupp.) can prevent isoflurane-induced caspase-3 activation and (memantine and topiramate). All animal experiments fol-apoptosis in vivo and in vitro. The results also indicated that lowed a protocol approved by the ethical committee on animalisoflurane-induced caspase activation and apoptosis are research at our institution. The neonatal HI brain damage wasdependent on cytosolic calcium levels (Zhang et al., 2008). In induced according to the modified Levine–Rice procedurerecent years, many studies focus on the protection of white (Northington, 2006; Rice et al., 1981; Vannucci and Vannucci,matter because the importance of PVL pathophysiology has 2005). For short, rat pups were anaesthetized by halothanebeen realized gradually (Khwaja and Volpe, 2008; Volpe, 2008). inhalation and duration of anesthesia was less than 5 min.NMDA receptor blockade with memantine acts as an effective The right common carotid artery was dissected, and doublypharmacological contributor with little side effects in attenu- ligated. One hour later, rats were then placed in a plasticating white matter injury, and the protective dose of chamber (37 °C) and exposed to 8% oxygen and 92% nitrogenmemantine does not affect normal myelination or cortical for 2 h. After this hypoxic exposure, the pups were returned togrowth (Manning et al., 2008; Micu et al., 2006). In our their dams for 2 h recovery.experiment, the apoptosis in the subcortical white matterwas reduced significantly in the combination group, which is 4.2. Drug administrationconsistent with the previous findings on caspase-3 activation. The present study demonstrated that a synergistic reduc- During recovery from HI, drugs were injected intraperitone-tion in brain damage could be achieved by combination of ally: vehicle group received vehicle (0.5 ml 0.9% saline)neuroprotective agents targeting different mechanisms. immediately after HI; memantine group received 20 mg/kgAlthough an evolving body of work has shown that combina- loading dose immediately after HI, then 1 mg/kg maintenancetion therapy holds promise in the treatment of HIBI, there has dose at 12 h intervals for 48h; topiramate group receivedbeen relatively little research on the combination therapy of 40 mg/kg loading dose then 10 mg/kg maintenance dose ontwo glutamate receptor antagonists. The combination of the same schedule as memantine; combination groupNMDA receptor antagonist MK-801 and AMPA receptor received both memantine and topiramate, the drug dosesantagonist NBQX shows an “overadditive” effect in cell culture and schedule were the same as above.and focal ischemia model in mice (Lippert et al., 1994). On theother hand, several studies on memantine or topiramate have 4.3. Gross brain damage gradingshown multidrug strategies are required for optimal thera-peutic outcome. The combination of memantine and clenbu- To quantify the severity of brain damage, rat pups wereterol not only reduces the infarct size but also extends the decapitated at 22d after HI and their brains were rapidlytherapeutic window of clenbuterol up to 2 h after ischemia dissected and frozen (Uhm et al., 2003). Then brains were(Culmsee et al., 2004). The combination of memantine and scored normal, mild, moderate or severe by a blinded observercelecoxib shows better effects in neuroprotection and anti- according to the method of Palmer et al. (1990). The neurologicinflammation in intracerebral hemorrhage treatment (Sinn et damage scores were given according to the following criteria.al., 2007). Combined treatment with topiramate and delayed Normal (1) is no reduction in the size of the right hemisphere,hypothermia improves both performance and pathological mild (2) is visible reduction in right hemisphere size, moderateoutcome in P15 and P35 rats (Liu et al., 2004b). (3) is large reduction in hemisphere size from a visible infarct Although the present study demonstrates the neuroprotec- in the right parietal area and severe (4) is near total destructiontive effect of memantine combined with topiramate, further of the hemisphere.studies are still needed in two aspects. A full dose–response To measure the loss of hemispheric weight, the brain wasexperiment was not performed in the present study, so further divided into two hemispheres and weighed after removing theinvestigation is still needed to determine the most optimal cerebellum and brainstem. Results are presented as thedosing regimen of memantine and topiramate. Noh et al. percent loss of hemispheric weight of the right side relativesuggested that the pretreatment with topiramate before HI to the left [(left − right) / left × 100]. The HI model used in thiswas more effective than the post-treatment after HI (Noh et al., study results in brain damage only on the ipsilateral side, thus2006). The result implies that the pretreatment with topiramate the loss of hemispheric weight can be used as a measure ofin the combination therapy can be considered in the future. brain damage in this model (Rice et al., 1981). Because the Collectively, the present study not only shows a promising brain weighs approximately 1 g/ml, weight loss is equivalenttherapy for neuroprotection, but also proposes a new para- to volume loss. According to the method by McDonald et al.,digm for multidrug development which is thought to be a the loss of brain weight on the ipsilateral side relative to thepromising approach in the treatment of HIBI. contralateral side is highly correlated with cellular damage (McDonald et al., 1989b). For short, weighing can assess the degree of brain damage.4. Experimental procedures 4.4. Microscopic brain damage grading4.1. Animal procedures Microscopic examination of the tissues was carried out toSeven-day-old rat pups of either sex, weighing between 12 g verify that the gross changes were a reflection of the expectedand 16 g, were used in this study. The rat pups were randomly histopathologic changes. The rat pups were anesthetized
    • 180 BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –18 2with pentobarbital 3 days after injury. Their brains wereperfusion fixed by cardiac puncture. They were flushed with Acknowledgmentssaline then fixed with 10% buffered formalin. After removal,the brains were stored in 10% buffered formalin. Sections This work was partly supported by Natural Science Founda-were then embedded with paraffin. Five-micron coronal tion of Guangdong Province, China. We gratefully thanksections were cut in the parietal region aiming for the Tianhua, Huang for his technical assistance.equivalent of Bregma −4.3 to −4.5 mm in the adult rat (Krugeret al., 1995) and then stained with hemotoxylin and eosin. REFERENCESCerebral cortex, hippocampus, striatum, thalamus wasscored from 0 to 5 by an observer blind to the treatment Andine, P., Thordstein, M., Kjellmer, I., Nordborg, C., Thiringer, K.,according to the method of Cataltepe et al. (1995), where “0” is Wennberg, E., Hagberg, H., 1990. Evaluation of brain damage innormal, “1” is 1–5% of neurons damaged, “2” is 6 to 25% of a rat model of neonatal hypoxic-ischemia. J. Neurosci. Methodsneurons damaged, “3” is 26–50% of neurons damaged, “4” is 35, 253–260.51–75% of neurons damaged, “5” is > 75% of neurons Barth, T.M., Stanfield, B.B., 1990. The recovery of forelimb-placingdamaged. behavior in rats with neonatal unilateral cortical damage involves the remaining hemisphere. J. Neurosci. 10, 3449–3459. Bittigau, P., Sifringer, M., Genz, K., Reith, E., Pospischil, D.,4.5. Neurofunctional assessment: foot-fault test Govindarajalu, S., Dzietko, M., Pesditschek, S., Mai, I., Dikranian, K., Olney, J.W., Ikonomidou, C., 2002. AntiepilepticThe foot-fault test was performed at 21d post-HI according to drugs and apoptotic neurodegeneration in the developinga published method (Bona et al., 1997). Rats were placed on brain. Proc. Natl. Acad. Sci. U. S. A. 99, 15089–15094.an elevated stainless steel grid floor 50 × 40 cm, 1 m above Block, F., Schwarz, M., 1996. Memantine reduces functional andthe floor with 3 cm2 holes and a wire diameter of 0.4 cm. morphological consequences induced by global ischemia in rats. Neurosci. Lett. 208, 41–44.Each pup was placed on the grid and observed for 2 min. The Bona, E., Johansson, B.B., Hagberg, H., 1997. Sensorimotor functionfoot-fault was defined as when the animal misplaced a fore- and neuropathology five to six weeks after hypoxia–ischemiaor hindlimb and the paw fell through between the grid bars. in seven-day-old rats. Pediatr. Res. 42, 678–683.The excess of left (contralateral foot-faults) to right (ipsilat- Cataltepe, O., Vannucci, R.C., Heitjan, D.F., Towfighi, J., 1995. Effecteral foot-faults) was recorded. Only the side difference of of status epilepticus on hypoxic–ischemic brain damage in thefoot-faults was used for the statistical evaluation to elim- immature rat. Pediatr. Res. 38, 251–257.inate the influence of the extent of activity in different rats Cha, B.H., Silveira, D.C., Liu, X., Hu, Y., Holmes, G.L., 2002. Effect of topiramate following recurrent and prolonged seizures during(Barth and Stanfield, 1990). early development. Epilepsy Res. 51, 217–232. Chen, H.S., Lipton, S.A., 2005. Pharmacological implications of two4.6. TUNEL staining and apoptotic cell counting distinct mechanisms of interaction of memantine with N-methyl-D-aspartate-gated channels. J. Pharmacol. Exp. Ther.We applied the Terminal deoxynucleotidyl transferase- 314, 961–971.mediated dUTP Nick End Labeling (TUNEL) staining to Chen, H.S., Pellegrini, J.W., Aggarwal, S.K., Lei, S.Z., Warach, S., Jensen, F.E., Lipton, S.A., 1992. Open-channel block ofdetect drug-induced apoptosis at 48 h after HI. All proce- N-methyl-D-aspartate (NMDA) responses by memantine:dures were performed following the manufacturers instruc- therapeutic advantage against NMDA receptor-mediatedtions (In Situ Cell Apoptosis Detection Kit I, POD; Boster, neurotoxicity. J. Neurosci. 12, 4427–4436.Wuhan, China). Chen, H.S., Wang, Y.F., Rayudu, P.V., Edgecomb, P., Neill, J.C., Segal, Cell counting was performed in the cortex, hippocampus, M.M., Lipton, S.A., Jensen, F.E., 1998. Neuroprotectivestriatum and the subcortical white matter. The hippocampus concentrations of the N-methyl-D-aspartate open-channelwas divided into the CA1, CA3 and dentate gyrus subfields. blocker memantine are effective without cytoplasmic vacuolation following post-ischemic administration and doPositive cells were counted at 400× magnification (one visual not block maze learning or long-term potentiation.field = 0.196 mm2). By use of the ImageJ software, all analyses Neuroscience 86, 1121–1132.were done by an individual who was unaware of treatment Culmsee, C., Junker, V., Kremers, W., Thal, S., Plesnila, N.,conditions. The average number of TUNEL-positive cells was Krieglstein, J., 2004. Combination therapy in ischemic stroke:calculated from at least three sections within each region for synergistic neuroprotective effects of memantine andeach animal. In the hippocampus subfields, counting was clenbuterol. Stroke 35, 1197–1202.performed throughout the entire region. In the cortex, Daval, J.L., Pourie, G., Grojean, S., Lievre, V., Strazielle, C., Blaise, S., Vert, P., 2004. Neonatal hypoxia triggers transient apoptosisstriatum and the subcortical white matter, three visual fields followed by neurogenesis in the rat CA1 hippocampus. Pediatr.were counted as average number per visual field. Only the Res. 55, 561–567.densely stained cells were counted as TUNEL-positive, de Lima, J., Beggs, S., Howard, R., 2000. Neural toxicity of ketamineslightly TUNEL-stained cells were not (Zhu et al., 2004). and othe NMDA antagonists. Pain 88, 311–312. Endres, M., Namura, S., Shimizu-Sasamata, M., Waeber, C., Zhang,4.7. Statistical analysis L., Gomez-Isla, T., Hyman, B.T., Moskowitz, M.A., 1998. Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J. Cereb.Data are presented as mean ± S.D., if not otherwise indicated. Blood Flow Metab. 18, 238–247.Comparisons were performed by one-way ANOVA with Fishers Feng, Y., Fratkins, J.D., LeBlanc, M.H., 2005. Estrogen attenuatespost hoc test. Differences were considered significant when hypoxic–ischemic brain injury in neonatal rats. Eur. J.p < 0.05. Pharmacol. 507, 77–86.
    • BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –1 82 181Feng, Y., Bhatt, A.J., Fratkin, J.D., Rhodes, P.G., 2008. Lu, Y.M., Yin, H.Z., Chiang, J., Weiss, J.H., 1996. Ca2+-permeable Neuroprotective effects of sodium orthovanadate after AMPA/kainate and NMDA channels: high rate of Ca2+ influx hypoxic–ischemic brain injury in neonatal rats. Brain Res. Bull. underlies potent induction of injury. J. Neurosci. 16, 5457–5465. 76, 102–108. Manning, S.M., Talos, D.M., Zhou, C., Selip, D.B., Park, H.K., Park,Follett, P.L., Deng, W., Dai, W., Talos, D.M., Massillon, L.J., C.J., Volpe, J.J., Jensen, F.E., 2008. NMDA receptor blockade with Rosenberg, P.A., Volpe, J.J., Jensen, F.E., 2004. Glutamate memantine attenuates white matter injury in a rat model of receptor-mediated oligodendrocyte toxicity in periventricular periventricular leukomalacia. J. Neurosci. 28, 6670–6678. leukomalacia: a protective role for topiramate. J. Neurosci. 24, McDonald, J.W., Roeser, N.F., Silverstein, F.S., Johnston, M.V., 4412–4420. 1989a. Quantitative assessment of neuroprotection againstGibbs III, J.W., Sombati, S., DeLorenzo, R.J., Coulter, D.A., 2000. NMDA-induced brain injury. Exp. Neurol. 106, 289–296. Cellular actions of topiramate: blockade of kainate-evoked McDonald, J.W., Silverstein, F.S., Johnston, M.V., 1989b. inward currents in cultured hippocampal neurons. Epilepsia 41 Neuroprotective effects of MK-801, TCP, PCP and CPP against (Suppl 1), S10–S16. N-methyl-D-aspartate induced neurotoxicity in an in vivoGlier, C., Dzietko, M., Bittigau, P., Jarosz, B., Korobowicz, E., perinatal rat model. Brain Res. 490, 33–40. Ikonomidou, C., 2004. Therapeutic doses of topiramate are not Mellon, R.D., Simone, A.F., Rappaport, B.A., 2007. Use of anesthetic toxic to the developing rat brain. Exp. Neurol. 187, 403–409. agents in neonates and young children. Anesth. Analg. 104,Grafe, M.R., 1994. Developmental changes in the sensitivity of the 509–520. neonatal rat brain to hypoxic/ischemic injury. Brain Res. 653, Micu, I., Jiang, Q., Coderre, E., Ridsdale, A., Zhang, L., Woulfe, J., Yin, 161–166. X., Trapp, B.D., McRory, J.E., Rehak, R., Zamponi, G.W., Wang,Haberny, K.A., Paule, M.G., Scallet, A.C., Sistare, F.D., Lester, D.S., W., Stys, P.K., 2006. NMDA receptors mediate calcium Hanig, J.P., Slikker Jr, W., 2002. Ontogeny of the accumulation in myelin during chemical ischaemia. N-methyl-D-aspartate (NMDA) receptor system and Nature 439, 988–992. susceptibility to neurotoxicity. Toxicol. Sci. 68, 9–17. Nakajima, W., Ishida, A., Lange, M.S., Gabrielson, K.L., Wilson,Hesselink, M.B., De Boer, B.G., Breimer, D.D., Danysz, W., 1999. M.A., Martin, L.J., Blue, M.E., Johnston, M.V., 2000. Apoptosis Brain penetration and in vivo recovery of NMDA receptor has a prolonged role in the neurodegeneration after hypoxic antagonists amantadine and memantine: a quantitative ischemia in the newborn rat. J. Neurosci. 20, 7994–8004. microdialysis study. Pharm. Res. 16, 637–642. Noh, M.R., Kim, S.K., Sun, W., Park, S.K., Choi, H.C., Lim, J.H., Kim,Ikonomidou, C., Bosch, F., Miksa, M., Bittigau, P., Vockler, J., I.H., Kim, H.J., Kim, H., Eun, B.L., 2006. Neuroprotective effect of Dikranian, K., Tenkova, T.I., Stefovska, V., Turski, L., Olney, topiramate on hypoxic ischemic brain injury in neonatal rats. J.W., 1999. Blockade of NMDA receptors and apoptotic Exp. Neurol. 201, 470–478. neurodegeneration in the developing brain. Science 283, Northington, F.J., 2006. Brief update on animal models of 70–74. hypoxic–ischemic encephalopathy and neonatal stroke.Johnson, J.W., Kotermanski, S.E., 2006. Mechanism of action of ILAR J. 47, 32–38. memantine. Curr. Opin. Pharmacol. 6, 61–67. Northington, F.J., Ferriero, D.M., Graham, E.M., Traystman, R.J.,Khwaja, O., Volpe, J.J., 2008. Pathogenesis of cerebral white matter Martin, L.J., 2001. Early neurodegeneration after injury of prematurity. Arch. Dis. Child., Fetal Neonatal Ed. 93, hypoxia–ischemia in neonatal rat is necrosis while delayed F153–F161. neuronal death is apoptosis. Neurobiol. Dis. 8, 207–219.Koh, S., Tibayan, F.D., Simpson, J.N., Jensen, F.E., 2004. NBQX or Palmer, C., Vannucci, R.C., Towfighi, J., 1990. Reduction of topiramate treatment after perinatal hypoxia-induced seizures perinatal hypoxic–ischemic brain damage with allopurinol. prevents later increases in seizure-induced neuronal injury. Pediatr. Res. 27, 332–336. Epilepsia 45, 569–575. Pappalardo, A., Liberto, A., Patti, F., Reggio, A., 2004.Kohr, G., 2007. NMDA receptor antagonists: tools in neuroscience [Neuroprotective effects of topiramate]. Clin. Ter. 155, 75–78. with promise for treating CNS pathologies. J. Physiol. 581, 1–2. Park, H.J., Kim, H.J., Ra, J., Zheng, L.T., Yim, S.V., Chung, J.H., 2008.Kruger, L., Saporta, S., Swanson, L.W., 1995. Photographic Atlas of Protective effect of topiramate on kainic acid-induced cell the Rat Brain. Cambridge University Press, Cambridge, UK. death in mice hippocampus. Epilepsia 49, 163–167.Lee, S.R., Kim, S.P., Kim, J.E., 2000. Protective effect of topiramate Parsons, C.G., Danysz, W., Quack, G., 1999. Memantine is a against hippocampal neuronal damage after global ischemia in clinically well tolerated N-methyl-D-aspartate (NMDA) receptor the gerbils. Neurosci. Lett. 281, 183–186. antagonist—a review of preclinical data. NeuropharmacologyLiao, D., Scannevin, R.H., Huganir, R., 2001. Activation of silent 38, 735–767. synapses by rapid activity-dependent synaptic recruitment of Poulsen, C.F., Schousboe, I., Sarup, A., White, H.S., Schousboe, A., AMPA receptors. J. Neurosci. 21, 6008–6017. 2006. Effect of topiramate and dBcAMP on expression of theLippert, K., Welsch, M., Krieglstein, J., 1994. Over-additive glutamate transporters GLAST and GLT-1 in astrocytes protective effect of dizocilpine and NBQX against neuronal cultured separately, or together with neurons. Neurochem. Int. damage. Eur. J. Pharmacol. 253, 207–213. 48, 657–661.Lipton, S.A., 2004. Failures and successes of NMDA receptor Puka-Sundvall, M., Hallin, U., Zhu, C., Wang, X., Karlsson, J.O., antagonists: molecular basis for the use of open-channel Blomgren, K., Hagberg, H., 2000. NMDA blockade attenuates blockers like memantine in the treatment of acute and chronic caspase-3 activation and DNA fragmentation after neonatal neurologic insults. NeuroRx 1, 101–110. hypoxia–ischemia. Neuroreport 11, 2833–2836.Lipton, S.A., 2006. Paradigm shift in neuroprotection by NMDA Rice III, J.E., Vannucci, R.C., Brierley, J.B., 1981. The influence of receptor blockade: memantine and beyond. Nat. Rev. Drug immaturity on hypoxic–ischemic brain damage in the rat. Ann. Discov. 5, 160–170. Neurol. 9, 131–141.Liu, L., Wong, T.P., Pozza, M.F., Lingenhoehl, K., Wang, Y., Sheng, Rogalewski, A., Schneider, A., Ringelstein, E.B., Schabitz, W.R., M., Auberson, Y.P., Wang, Y.T., 2004a. Role of NMDA receptor 2006. Toward a multimodal neuroprotective treatment of subtypes in governing the direction of hippocampal synaptic stroke. Stroke 37, 1129–1136. plasticity. Science 304, 1021–1024. Rosi, S., Vazdarjanova, A., Ramirez-Amaya, V., Worley, P.F.,Liu, Y., Barks, J.D., Xu, G., Silverstein, F.S., 2004b. Topiramate Barnes, C.A., Wenk, G.L., 2006. Memantine protects against extends the therapeutic window for hypothermia-mediated LPS-induced neuroinflammation, restores neuroprotection after stroke in neonatal rats. Stroke 35, behaviorally-induced gene expression and spatial learning in 1460–1465. the rat. Neuroscience 142, 1303–1315.
    • 182 BR A I N R ES E A RC H 1 2 8 2 ( 2 00 9 ) 1 7 3 –18 2Schubert, S., Brandl, U., Brodhun, M., Ulrich, C., Spaltmann, J., Vannucci, R.C., Vannucci, S.J., 2005. Perinatal hypoxic–ischemic Fiedler, N., Bauer, R., 2005. Neuroprotective effects of brain damage: evolution of an animal model. Dev. Neurosci. 27, topiramate after hypoxia–ischemia in newborn piglets. 81–86. Brain Res. 1058, 129–136. Villmann, C., Becker, C.M., 2007. On the hypes and falls inSfaello, I., Baud, O., Arzimanoglou, A., Gressens, P., 2005. neuroprotection: targeting the NMDA receptor. Neuroscientist Topiramate prevents excitotoxic damage in the newborn 13, 594–615. rodent brain. Neurobiol. Dis. 20, 837–848. Volpe, J.J., 2008. Neurology of the Newborn, 5th edn. Elsevier,Sinn, D.I., Lee, S.T., Chu, K., Jung, K.H., Song, E.C., Kim, J.M., Philadelphia. Park, D.K., Kim, M., Roh, J.K., 2007. Combined neuroprotective Zhang, G., Dong, Y., Zhang, B., Ichinose, F., Wu, X., Culley, D.J., effects of celecoxib and memantine in experimental Crosby, G., Tanzi, R.E., Xie, Z., 2008. Isoflurane-induced intracerebral hemorrhage. Neurosci. Lett. 411, caspase-3 activation is dependent on cytosolic calcium and 238–242. can be attenuated by memantine. J. Neurosci. 28, 4551–4560.Uhm, C.S., Kim, K.B., Lim, J.H., Pee, D.H., Kim, Y.H., Kim, H., Eun, Zhu, C., Wang, X., Cheng, X., Qiu, L., Xu, F., Simbruner, G., B.L., Tockgo, Y.C., 2003. Effective treatment with fucoidin for Blomgren, K., 2004. Post-ischemic hypothermia-induced tissue perinatal hypoxic–ischemic encephalopathy in rats. Neurosci. protection and diminished apoptosis after neonatal cerebral Lett. 353, 21–24. hypoxia–ischemia. Brain Res. 996, 67–75.
    • ARTICLES Neuronal glutathione deficiency and age-dependent© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience neurodegeneration in the EAAC1 deficient mouse Koji Aoyama1,2, Sang Won Suh1,2, Aaron M Hamby1,2, Jialing Liu2,3, Wai Yee Chan1,2, Yongmei Chen1,2 & Raymond A Swanson1,2 Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier–1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically EAAC1-null (Slc1a1–/–) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in the hippocampal slices of EAAC1–/– mice were found to have reduced glutathione content, increased oxidant levels and increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1–/– mice with N-acetylcysteine, a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and age-dependent neurodegeneration. Sodium-dependent excitatory amino acid transporters (EAATs) regu- in neurons17 and extremely low (o 300 nM) cystine concentrations in late extracellular glutamate concentrations in the central nervous brain extracellular fluid18,19. Neuronal glutathione synthesis is sup- system. Five EAATs have been identified, termed glutamate-aspartate ported by astrocytes through an indirect route involving astrocyte transporter (GLAST or EAAT1), glutamate transporter 1 (GLT-1 or glutathione release, its cleavage to cysteinylglycine and the subsequent EAAT2), EAAC1 (EAAT3), EAAT4 and EAAT5 (ref. 1). GLAST and release of free cysteine by an ectopeptidase located on the neuronal cell GLT-1 are localized primarily to astrocytes, whereas EAAC1, EAAT4 and surface13,20. The route of cysteine uptake into neurons has not been EAAT5 are localized primarily to neurons1–4. EAAT4 and EAAT5 are ascertained, but cell culture studies suggest EAAC1 as a candidate restricted to cerebellar Purkinje cells and retina, respectively, but EAAC1 neuronal cysteine transporter because pharmacological inhibitors of is widely expressed in neurons throughout the nervous system3,4. EAAC1 prevent neuronal glutathione synthesis in the presence of The function of EAAC1 in the brain has not been established. Unlike extracellular cysteine14,21,22. Cysteine is transported by EAAC1 at a the astrocyte glutamate transporters, EAAC1 does not play a major role rate comparable to that of glutamate and with an affinity roughly in clearing glutamate from the extracellular space5–7. Also unlike the tenfold greater than that of the astrocyte transporters GLAST and astrocyte glutamate transporters, which are clustered near glutamater- GLT-1 (ref. 10). gic synapses, EAAC1 is localized diffusely over cell bodies and pro- Glutathione is important for the metabolism of hydrogen peroxide cesses2,3,8, suggesting a function other than the re-uptake of (H2O2), nitric oxide and other reactive oxygen species and for the synaptically released glutamate. An exception to this pattern is pre- maintenance of reduced thiol groups on proteins23. Pharmacologically synaptic GABAergic terminals, where EAAC1 uptake of glutamate induced glutathione deficiency causes neurodegeneration24, and low- contributes to re-synthesis of GABA9. An additional distinguishing ered glutathione content is found in neurodegenerative disorders feature of EAAC1 is that it can bind and transport cysteine far more associated with oxidative stress25, suggesting that impairment in effectively than the astrocyte glutamate transporters can10,11. neuronal cysteine uptake could lead to neurodegeneration. Here we Cysteine is normally the rate-limiting substrate for the synthesis of report that mice deficient in EAAC1 are deficient in neuronal thiol glutathione, the principal cellular thiol antioxidant12–14. Most cell types content and develop age-dependent behavioral abnormalities and brain acquire cysteine in the form of cystine, by hetero-exchange with atrophy. Neurons in hippocampal slices from EAAC1–/– mice showed glutamate (system Xc–)12, but cell culture studies suggest that neurons increased vulnerability to oxidants (but not to glutamate) and reduced lose this capacity during development13,15,16. Consistent with this, capacity to metabolize reactive oxygen species. Neuronal thiol content studies of the intact mature brain show an absence of Xc– expression and resistance to oxidant stress were normalized in the EAAC1–/– mice 1Department of Neurology, University of California San Francisco, San Francisco, California 94143, USA. 2Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, California 94121, USA. 3Department of Neurosurgery, University of California San Francisco, San Francisco, California 94143, USA. Correspondence should be addressed to R.A.S. (ray@itsa.ucsf.edu). Received 13 September; accepted 28 October; published online 27 November 2005; doi:10.1038/nn1609 NATURE NEUROSCIENCE VOLUME 9 [ NUMBER 1 [ JANUARY 2006 119
    • ARTICLES Figure 1 Genotyping and glutamate transporter expression. (a) PCR analysis a b – –/ e of genomic DNA shows loss of the EAAC1 band and presence of the NEO p 1 ty AC ild (kDa) cassette in the outbred EAAC1–/– mouse. (b) Western blots show the major EA W EAAC1 band at 63 kDa in the wild-type brain and no immunoreactivity in the – –/ pe 1 ty EAAC1–/– mouse brain. (c) Immunostaining of the hippocampal CA1 region AC ild 170.0 EA W 115.5 shows EAAC1 expression localized to neuronal cell membranes in the wild- EAAC1 82.2 EAAC1 type brain and no signal from the EAAC1–/– mouse brain. Scale bar, 40 mm. Neo (d) Western blots for brain GLT-1 and GLAST expression in 11-month-old 64.2 wild-type and EAAC1–/– mice. 48.8© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience β-actin Behavioral abnormalities in the EAAC1–/– mice c Wild type EAAC1 –/– In maintaining the EAAC1–/– mouse colonies, it became apparent that the older mice showed increased aggressiveness and impaired self- grooming compared to age-matched wild-type mice. We studied their behavioral changes further with the Morris water maze test27. The performance of the wild-type and EAAC1–/– mice was similar at 7 weeks of age, showing progressively shortened target latency with d GLAST repeated trials on both the visible platform and the hidden platform tasks. By contrast, 11-month-old EAAC1–/– mice did not improve on β-actin either task with repeated trials (Fig. 2a). This impairment was not due GLT1 to gross visual disturbances, because the aged EAAC1–/– mice reached normally for nearby small surfaces when suspended by the tail. β-actin Spontaneous locomotor activity was also not significantly altered in the aged EAAC1–/– mice (data not shown). Spontaneous swim velocity was slower in the aged EAAC1–/– mice (Fig. 2b), but not slow enough to – – – – pe pe pe pe –/ –/ –/ –/ ty ty ty ty 1 1 1 1 AC AC AC AC ild ild ild ild account for the failure to shorten the target latency with repeated trials. W W W W EA EA EA EA Notably, the aged EAAC1–/– mice differed from the other groups in that their spontaneous swim speed was well below their maximal swim by the administration of N-acetylcysteine (NAC), a membrane- speed. Together, these observations suggest cognitive or motivational permeable cysteine precursor that does not require active transport. impairment in the aged EAAC1–/– mice. These findings suggest that EAAC1 functions as a neuronal cysteine transporter and that dysfunction of this system leads to impaired Brain atrophy and oxidative stress in EAAC1–/– mice glutathione homeostasis and neurodegeneration. A comparison of coronal sections from wild-type and EAAC1–/– mouse brains showed age-dependent cortical thinning and ventricular enlar- RESULTS gement in the EAAC1–/– mice. Wild-type mice showed a small increase We determined mouse genotype by polymerase chain reaction (PCR) in ventricular size between the ages of 7 weeks and 11 months (Fig. 3); and confirmed genotype results by western blotting and immunostain- by contrast, EAAC1–/– mice showed slightly larger ventricle size than ing for EAAC1 protein expression (Fig. 1). Western blots showed the wild-type mice at 7 weeks and much larger ventricle size at EAAC1 immunoreactivity at the predicted molecular weight 11 months. Accordingly, measures of the hippocampal CA1 cell layer (B63 kDa)2,26 in the brains of wild-type mice (Fig. 1b) and no and the corpus callosum both showed reduced size in the aged immunoreactivity in the brains of EAAC1–/– mice. Similarly, immu- EAAC1–/– mice (Fig. 3). nostaining of hippocampal sections showed that EAAC1 was expressed on neuronal cell membranes of wild-type but not EAAC1–/– mice (Fig. 1c). Previous studies have found no change in the expression of a Wild type EAAC1–/– the major astrocyte glutamate transporters GLT-1 and GLAST in Age 7 weeks Age 11 months response to EAAC1 gene deficiency6 or downregulation7, but expres- 60 60 sion of these transporters has not been examined in the aged EAAC1–/– 50 50 Latency (s) Latency (s) 40 40 mouse brain. Here, western blots for GLT-1 and GLAST showed no 30 30 difference between the wild-type mice and EAAC1–/– mice at either 20 20 7 weeks (data not shown) or 11 months of age (Fig. 1d), as determined 10 10 by densitometry. 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 Visible Hidden Visible Hidden Figure 2 Performance on the Morris water maze test. (a) At age 7 weeks, time to reach platform (latency) and rate of latency change was similar in the b Age 7 weeks Wild type EAAC1–/– Age 11 months wild-type and EAAC1–/– mice during both visible and hidden platform Swim velocity (cm s–1) Swim velocity (cm s–1) sessions. For both the visible and hidden platform tasks, the 11-month-old 30 30 EAAC1–/– mice showed profound impairment (P o 0.01) as compared to the 7-week- and 11-month-old wild-type mice and the 7-week-old EAAC1–/– mice 20 20 (n ¼ 10). The dashed and dotted lines indicate the mean rate of change 10 10 over the designated testing intervals. (b) Spontaneous swim velocity was moderately reduced in the 11-month-old EAAC1–/– mice relative to the 0 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 1 Day 2 Day 3 Day 4 Day 5 7-week-old EAAC1–/– and wild-type mice and the 11-month-old wild-type mice (P o 0.01, n ¼ 10). Visible Hidden Visible Hidden 120 VOLUME 9 [ NUMBER 1 [ JANUARY 2006 NATURE NEUROSCIENCE
    • ARTICLES ** a b c 2 Wild type Ventricle area (mm2) Age 7 weeks Age 11 months Age 7 weeks Age 11 months EAAC1 –/– Wild type EAAC1 –/– Wild type EAAC1 –/– Wild type EAAC1 –/– Wild type EAAC1 –/– 1.5 ** 1 * * 0.5 0 ks s ks s th th ee ee on on w w m m 7 7 11 11© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience Anterior Posterior d Age 7 weeks e Age 7 weeks Age 11 months f 200 Wild type ** Structure width (µm) Wild type EAAC1 –/– Wild type EAAC1 –/– Wild type EAAC1 –/– EAAC1 –/– 150 Corpus * 100 callosum Age 11 months 50 –/– Wild type EAAC1 0 ks s ks s th th ee ee on on w w m m 7 7 11 11 CA1 Corpus callosum Figure 3 Brain atrophy in the EAAC1–/– mice. (a,b) Coronal brain sections show cortical thinning and ventricular enlargement in the older EAAC1–/– mice. The top rows are at the level of the anterior commissure and the bottom rows are 2.7 mm posterior to the anterior commissure. Scale bar, 2 mm in a; 1 mm in b. (c) Ventricle size is larger in the EAAC1–/– mice at age 7 weeks, and the difference is further increased at age 11 months (n ¼ 6–14). (d) Hematoxylin-eosin staining of the CA1 hippocampal cell field; scale bar, 40 mm. (e) Fluoro-myelin staining of the corpus callosum (green) in coronal section; scale bar, 100 mm. Nuclear counterstaining (red) of the pyramidal cell layer caught obliquely on these sections is included for scale and orientation. (f) Quantified measures of the CA1 cell layer and corpus callosum (n ¼ 3–6). *P o 0.05; **P o 0.01. Error bars denote s.e.m. The age-dependent brain atrophy in the EAAC1–/– mice was accom- Increased vulnerability of EAAC1–/– neurons to oxidants panied by markers of oxidative stress. Immunoreactivity for nitro- We prepared hippocampal slices from young (6–8 week) wild-type and tyrosine and 4-hydroxy-2-nonenal (HNE), which are formed by EAAC1–/– mice to study possible mechanisms by which EAAC1 gene oxidant interactions with proteins and lipids, respectively, was deficiency could lead to the observed age-dependent changes. Because increased in the aged EAAC1–/– mouse brains. The increase was EAAC1 can function as both a glutamate and a cysteine transporter10,11, prominent in the hippocampal cell fields and the cerebral cortex we used hippocampal slices to compare the vulnerability of the wild- (Fig. 4). Both nitrotyrosine and HNE localized to neurons in the type and EAAC1–/– mouse brains to glutamate and oxidant exposures. aged EAAC1–/– mouse brains (Fig. 4). These markers were not detected Neurons in slices from the EAAC1–/– mice did not show increased in the corpus callosum or other white matter tracts (Supplementary vulnerability to glutamate over a range of bath glutamate concentra- Fig. 1 online). tions (Fig. 5). This result is consistent with earlier studies reporting a c Nitrotyrosine MAP-2 Merge Nitrotyrosine 4-hydroxy-2-nonenal a Wild type EAAC1–/– b Wild type EAAC1–/– Nitrotyrosine GFAP Merge CA1 CA1 d CA3 HNE MAP-2 Merge CA3 Cortex Cortex HNE GFAP Merge Figure 4 Oxidative stress in neurons of EAAC1–/– mouse brain. (a,b) Immunostaining for (a) nitrotyrosine and (b) HNE showed increased immunoreactivity in neurons of cerebral cortex and hippocampal CA1 and CA3 cell fields in 11-month-old EAAC1–/– mice. Scale bar, 40 mm. (c,d) Immunostaining for nitrotyrosine and HNE, colocalized with the neuronal marker microtubule-associated protein-2 (MAP-2). Representative of three brains in each group. NATURE NEUROSCIENCE VOLUME 9 [ NUMBER 1 [ JANUARY 2006 121
    • ARTICLES Wild type EAAC1–/– Figure 5 Increased vulnerability of neurons in EAAC1–/– mice to oxidative a b stress. (a) Neuron death in hippocampal slice preparations was identified by PI fluorescence. PI fluorescence was evaluated at 0 or 4 h under control control 0h conditions, or 3.5 h after a 30-min incubation with glutamate, hydrogen peroxide or SIN-1; scale bar, 40 mm. (b) There was a severalfold increased Wild type sensitivity to SIN-1 and H2O2 in the slices from EAAC1–/– mice, but no ** increased sensitivity to glutamate. **P o 0.01; n ¼ 3–5. Error bars control 4h 30 EAAC1–/– represent s.e.m. Fluorescence intensity 25 ** (arbitrary units) H2O2 200 µM glutamate 10 mM 20 also showed increased nitrotyrosine formation (Fig. 6a,b), supporting© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience 15 the possibility that EAAC1 gene deficiency leads to impaired neuronal 4h 10 glutathione synthesis. To further assess this possibility, hippocampal 5 slices from EAAC1–/–, wild-type and BSO-treated wild-type mice were 0 evaluated with 5-(and 6-)carboxy-2¢,7¢-dichlorodihydrofluorescein l l 4 Glu M lu mM SI 2 20 M µM 50 M ro tro 4h diacetate (DCF), which is oxidized to a fluorescent compound by h 5m 4 2O 0 m -1 0 µ nt G on 0 co 5 c 2. 1 oxygen species derived from H2O2 or peroxynitrite33. We observed a h h lu 0 4 G N h H h 4 modest increase in neuronal DCF fluorescence in the wild-type slices 4 h SIN-1 500 µM h 4 during incubation with H2O2 or SIN-1 and much larger increases (10- 4h to 40-fold) in the slices from EAAC1–/– mice and BSO-treated wild-type mice (Fig. 6c,d). The effect of BSO pretreatment was slightly less than the effect of the EAAC1–/– genotype. negligible role for neuronal glutamate transporters in regulating extra- We measured bulk glutathione content in brain homogenates from cellular glutamate concentrations1,9. By contrast, the neurons in EAAC1–/–, wild-type and BSO-treated wild-type mice (Fig. 6e). These EAAC1–/– mice were several times more sensitive to H2O2 and to measurements are likely to underestimate the degree of glutathione 3-morpholinosydnonimine (SIN-1), which generates superoxide, nitric deficiency in the EAAC1–/– neurons because a large share of brain oxide, peroxynitrite and related oxygen species28. glutathione is localized to astrocytes, which do not express EAAC1 Cysteine uptake is a rate-limiting step in neuronal synthesis of (ref. 23). Similarly, because the decrease in glutathione in the BSO- glutathione21,23,29, and glutathione has a central role in the metabolism treated mice reflects impaired glutathione synthesis in both neurons of both peroxide and nitrosyl oxidants23,30,31. SIN-1 greatly increased and astrocytes, the comparable glutathione reductions observed in the neuronal nitrotyrosine immunoreactivity in brain slices from EAAC1–/– mouse brains and BSO-treated mouse brains may indicate a EAAC1–/– mice under conditions that produced a negligible increase much greater neuronal glutathione depletion in the EAAC1–/– mice. in slices from the wild-type mice (Fig. 6a,b), suggesting an impaired Glutathione measurements from the livers of wild-type and EAAC1–/– capacity for the scavenging of nitrosyl radicals in the EAAC1–/– mouse mice gave comparable values (mean ± s.e.m.)—1.60 ± 0.12 and 1.50 ± brain31. We performed parallel studies with hippocampal slices from 0.06 mmol per mg of protein, respectively (n ¼ 4)—suggesting that the wild-type mice that had been treated with buthionine sulfoximine reduced glutathione levels in the EAAC1–/– mouse brains results from (BSO) to reduce brain glutathione content32. Slices from these mice local rather than systemic effects of EAAC1 gene deficiency. a Wild type EAAC1 –/– Wild type + BSO b c Wild type EAAC1–/– Wild type + BSO Nitrotyrosine ** 0 min 50 30 min control Wild type ** control Arbitrary density 40 Wild type + BSO –/– 30 EAAC1 30 min 20 30 min control 10 H2O2 200 µM 0 l l µM ro tro nt n 0 co co 50 30 min 30 min in in -1 SIN-1 500 µM SIN-1 500 µM m m N 0 30 SI in m 30 Figure 6 Reduced scavenging of reactive oxygen species in neurons of EAAC1–/– mice. d DCF fluorescence e (a) Hippocampal slices were prepared from EAAC1–/– mice, wild-type mice or wild-type mice ** that had been treated with BSO to reduce brain glutathione content. The slices were evaluated ** ** (µmol per mg protein) 60 Wild type for nitrotyrosine immunoreactivity after incubation with SIN-1; scale bar, 40 mm. (b) SIN-1 ** Wild type Arbitrary density 1.0 produced a small increase in nitrotyrosine immunoreactivity in the wild-type brain slices + BSO 0.8 40 ** ** GSH –/– 0.6 (P o 0.01) and a much larger increase in slices from EAAC1–/– mice and BSO-treated wild- EAAC1 0.4 type mice (**P o 0.01, n ¼ 4). (c) The presence of reactive oxygen species was evaluated 20 0.2 with DCF after 30-min incubations with H2O2 or SIN-1; bar, 40 mm. (d) Both of the oxidants 0 – ild AA ype O –/ BS produced a small increase in the neuronal DCF signal in the wild-type hippocampal slices 0 ty C1 t ild + (P o 0.01), and both oxidants produced much larger increases in slices from EAAC1–/– mice l l W µM µM pe ro ro E nt nt 0 0 co co and BSO-treated wild-type mice. (P o 0.01, n ¼ 4). (e) Brain glutathione content was reduced 20 50 in in W -1 2 m m 2O in the EAAC1–/– mouse brains and in wild-type mice treated with BSO (**P o 0.01, n ¼ 4–8). N 0 30 SI H in in Error bars denote s.e.m. m m 30 30 122 VOLUME 9 [ NUMBER 1 [ JANUARY 2006 NATURE NEUROSCIENCE
    • ARTICLES a H2O2 b SIN-1 c d Bic (–) Bic (+) Bic (–) Bic (+) H2O2 SIN-1 0 min 0 min control control 14 Bic (–) 14 Bic (–) Arbitrary density Arbitrary density 12 Bic (+) 12 Bic (+) 10 10 8 8 30 min 30 min 6 6 control control 4 4 2 2 0 0© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience 30 min 30 min µM l l M l l M µM ro ro ro ro SIN-1 500 µM m H2O2 200 µM m nt nt nt nt 0 00 co co 5 co co 2 1 50 -1 2 2 in in 2O in in -1 N m m m m 2O SI N H SI 0 30 0 30 H in in m in m in m 30 min 30 min m 30 30 30 30 H2O2 1 mM SIN-1 5 mM e H2O2 f SIN-1 g h Bic (–) Bic (+) Bic (–) Bic (+) 0h 0h H2O2 SIN-1 control control 12 12 Bic (–) Bic (–) Arbitrary density Arbitrary density 10 10 Bic (+) Bic (+) 8 8 4h 4h 6 6 control control 4 4 2 2 4h 4h 0 0 H2O2 200 µM SIN-1 500 µM l l M l l M µM µM ro ro ro ro m m nt nt nt nt 00 0 co co 2 1 co co 5 50 -1 2 2 2O h h h h N -1 0 4 2O 0 4 H SI N H SI h h 4h 4h 4 h 4 h 4 4 H2O2 1 mM SIN-1 5 mM Figure 7 Bicuculline does not potentiate the oxidant effects of H2O2 or SIN-1. (a–h) The presence of reactive oxygen species in wild-type hippocampal cultures was evaluated with DCF after 30-min incubations with H2O2 (a,c) or SIN-1 (b,d) in the presence and absence of 20 mM bicuculline. Neuron death in wild-type hippocampal slice preparations was assessed by PI fluorescence after incubation with H2O2 (e,g) or SIN-1 (f,h) in the presence and absence of 20 mM bicuculline. Scale bar, 40 mm; n ¼ 4 under each condition. Error bars denote s.e.m. Oxidant effects on neurons are unaffected by bicuculline normalized in EAAC1–/– mice given NAC 5 h before brain harvest The uptake of glutamate by EAAC1 provides a substrate for GABA (Fig. 8a). To confirm that this thiol signal was due to glutathione rather formation in GABAergic neurons9, raising the possibility that EAAC1 than to NAC or cysteine, we also prepared slices from EAAC1–/– mice deficiency could promote oxidant stress in neurons by reducing that were given BSO along with NAC to prevent de novo synthesis of GABAergic tone. GABA itself has no significant antioxidant properties, glutathione. The effect of NAC was blocked in these mice (Fig. 8a), but reduced activation of GABAA receptors could, in principle, confirming that the C5 maleimide signal is primarily attributable to indirectly amplify oxidant effects on neurons by increasing neuronal glutathione. Biochemical measures of glutathione further confirmed depolarization, NMDA receptor activation and glutamate release34,35. that the NAC-induced increase in brain glutathione content was The comparable neurotoxicity of glutamate in brain slices from wild- blocked by BSO and that the BSO treatment did not deplete pre- type and EAAC1–/– mice (Fig. 5) suggests that this effect, if present, existing glutathione stores over this time interval (Fig. 8b). As must be small; but to directly test this possibility, we examined the expected, neuronal death after oxidant exposure was decreased in slices effect of the GABAA receptor antagonist (+)-bicuculline on the from NAC-treated EAAC1–/– mice relative to untreated EAAC1–/– mice neuronal response to oxidants in the brain slice preparation. We (Figs. 5 and 8), and this decrease was negated by the coadministration observed no effect of 20 mM bicuculline36 on DCF fluorescence in of BSO (Fig. 8c,d). wild-type slices after incubation with SIN-1 or H2O2 (Fig. 7a–d). Bicuculline also had no effect on neuronal survival after incubation DISCUSSION with SIN-1 or H2O2 (Fig. 7e–h). The original description of the EAAC1–/– mouse reported reduced spontaneous activity but no gross neurodegeneration6. Our studies, NAC normalizes neuronal glutathione in EAAC1–/– mice using descendants of these mice, similarly showed no gross neuro- To determine directly whether EAAC1 is involved in neuronal glu- degeneration at young ages, but did show brain atrophy and pro- tathione homeostasis, we used fluorescently tagged C5 maleimide to nounced behavioral abnormalities by 11 months of age. These changes quantify reactive thiol content (of which glutathione is the principal were accompanied by histochemical markers of neuronal oxidative component) in hippocampal slices from wild-type and EAAC1–/– mice. stress, and experiments with acutely prepared hippocampal slices As expected, the C5 maleimide fluorescence was markedly reduced in confirmed an impaired neuronal resistance to reactive oxygen species. neurons from the hippocampal slices of EAAC1–/– mice (Fig. 8a). NAC The capacity of EAAC1 to function as a cysteine transporter raised the can passively cross lipid membranes and thereby provide cysteine to possibility that these abnormalities could be caused by impaired cells that lack cysteine transport37,38. The C5 maleimide signal was neuronal glutathione metabolism, and we confirmed decreased reactive NATURE NEUROSCIENCE VOLUME 9 [ NUMBER 1 [ JANUARY 2006 123
    • ARTICLES Wild type EAAC1–/– a b c EAAC1–/– EAAC1–/– NAC 2.0 NAC NAC + BSO NAC NAC + BSO BSO (µmol per mg protein) NAC + BSO 1.5 * Glutathione * 4 h H2O2 1.0 0h 200 µM EAAC1–/– control EAAC1–/– + NAC + NAC + BSO 0.5 4h 4 h SIN-1 0 0 4 6 control 500 µM© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience Time (h) Figure 8 Neuronal glutathione deficiency and vulnerability to oxidant stress in EAAC1–/– mice are both d 20 EAAC1–/– ** ** Fluorescence intensity + NAC reversed by NAC. (a) Reactive thiols were evaluated in brain slices using C5-maleimide fluorescence. There (arbitrary units) 15 EAAC1–/– was reduced fluorescence in the neurons in EAAC1–/– slices relative to those in the wild-type slices. This + NAC + BSO signal was increased in slices from mice treated with the cell-permeant cysteine precursor NAC, but not in 10 slices from mice treated with BSO together with NAC to prevent glutathione synthesis. n ¼ 3 in each group. (b) Biochemical determination of glutathione in EAAC1–/– mouse brains after treatment with NAC or 5 NAC plus BSO confirmed that the effect of NAC on glutathione levels is attenuated by the concomitant administration of BSO. *P o 0.05 versus control, n ¼ 3–7. (c,d) PI staining in hippocampal slices 0 l l µM µM prepared from NAC-treated EAAC1–/– mice showed increased neuronal resistance to H2O2 or SIN-1 ro ro nt nt 0 0 co co 20 50 compared to untreated EAAC1–/– mice (Fig. 5). This effect of NAC was blocked by the coadministration of h h -1 2 0 4 O BSO. **P o 0.01, n ¼ 3. Error bars denote s.e.m. N 2 SI H h h 4 4 thiol content in hippocampal neurons of the EAAC1–/– mice. The other interacting factors associated with aging or, alternatively, to the administration of the cell-permeable cysteine precursor NAC corrected accumulated effect of a prolonged impairment in neuronal glutathione both the neuronal thiol content and neuronal oxidant-scavenging homeostasis. In either case, the parallel between the delayed onset of capacity. The effects of NAC on oxidant-scavenging capacity are due disease in these mice and the delayed onset of most human neurode- primarily to its role as a substrate for glutathione synthesis39,40; we generative disorders suggests that common mechanisms could be confirmed this here by showing that the effects of NAC are negated by involved. There have been case reports of neurological developmental the simultaneous administration of BSO. delay associated with dicarboxylic aminoaciduria42, but a human Immunostaining showed nitrotyrosine and HNE accumulation in genetic EAAC1 deficiency has not been identified. EAAC1 expression neurons in the EAAC1–/– mouse brain, consistent with the localization is, however, regulated by several factors, including protein kinase C43, of EAAC1 selectively to neurons. The measured loss of neurons in the cholesterol44, presenilin26 and others. It will be of interest to learn hippocampal CA1 cell layer was small, however, relative to the whether the magnitude of these regulatory influences is sufficient to increased ventricular volume. This discrepancy may be explained in alter neuronal glutathione metabolism. part by associated axonal loss, as demonstrated by the narrowing of the corpus callosum in the aged EAAC1–/– mice. In addition, there may be METHODS secondary loss of glial elements contributing to brain atrophy. Studies were performed in accordance with a protocol approved by the Animal EAAC1 is unique among the cysteine transporters in that its Use Committee at the San Francisco Veterans Affairs Medical Center. Reagents transport of substrates is coupled to the transport of three Na+ ions, were obtained from Sigma-Aldrich except where noted. thereby enabling uptake against a steep concentration gradient41. The EAAC1–/– mice. The EAAC1–/– mice were descendants of the strain established present findings show that EAAC1 is quantitatively important for in a previous study6, in which exon 1 is disrupted by a neomycin resistance neuronal glutathione homeostasis but do not exclude alternative routes (NEO) cassette. These mice were outbred to wild-type CD-1 mice for six or of neuronal cysteine uptake that may partially compensate for EAAC1 more generations before these studies. Littermate and age-matched wild-type deficiency. In fact, a complete loss of cysteine uptake would not be CD-1 mice were used as controls. compatible with prolonged neuronal survival. Genotyping. Genotypes were confirmed by PCR of tail DNA, using oligonu- EAAC1 is also expressed in several extraneural tissues6. In the kidney, cleotide primers pairs for EAAC1 exon 1 and for the inserted NEO cassette: EAAC1 is expressed by renal tubule cells, in which it serves as a major 5¢-CCGCCACGCAAAACCACCGTGCTCGGTCCC-3¢ (EAAC1.1), 5¢-CTAG route of glutamate and aspartate re-uptake from the urine. EAAC1–/– TACCACGGCGGCCACGGTTGAGAGCA-3¢ (EAAC1.2), 5¢-CATTCGACCAC mice show dicarboxylic aminoaciduria6, and it is possible that this CAAGCGAAAC-3¢ (NEO.1) and 5¢-CAGCAATGTCACGGGTAGCCAAC-3¢ metabolic defect could influence neuronal glutathione metabolism (NEO.2). The amplified products were separated by 1.5% agarose gel electro- indirectly. However, the rapid normalization of neuronal glutathione phoresis and visualized by ethidium bromide staining. with NAC identifies cysteine uptake as the limiting step for glutathione synthesis in EAAC1–/– mice. Similarly, the normal glutathione content Western blotting. Western blotting was performed as described pre- measured in the liver of the EAAC1–/– mouse argues against a systemic viously45. Rabbit antibodies to EAAC1, GLAST or GLT-1 (Alpha Diagnostic International) were used at 0.5 mg ml–1, and mouse monoclonal antibody to deficiency of sulfur amino acids. b-actin (Sigma) was used at a 1:1,000 dilution. The antibodies were visualized A notable feature of the EAAC1–/– mouse is that the brain atrophy by chemiluminescence after incubation with horseradish peroxidase– and behavioral abnormalities are markedly age dependent, despite the conjugated antibody to rabbit IgG. Optical densities of the protein bands fact that brain slices obtained from young mice showed large biochem- were measured using the NIH ImageJ software program. The protein band ical abnormalities when compared to slices from wild-type mice. The densities were normalized in each case to the density of the b-actin band from delayed onset of gross atrophy and behavioral changes may be due to the same sample. 124 VOLUME 9 [ NUMBER 1 [ JANUARY 2006 NATURE NEUROSCIENCE
    • ARTICLES Immunostaining. Brain sections were prepared and immunostained as cence or fixed in 4% paraformaldehyde for nitrotyrosine immunostaining. DCF described45. The primary antibodies were 5 mg ml–1 rabbit antibody to EAAC1 fluorescence and nitrotyrosine immunofluorescence was quantified by the same (Alpha Diagnostic International); 1:500 dilution rabbit antibody to nitro- method as used for PI fluorescence. tyrosine (Chemicon); and 1:500 dilution rabbit antibody to HNE (Alpha Diagnostic International). After washing, the slices ware incubated with a Manipulation and measurement of glutathione level. BSO was injected at a 1:500 dilution of Alexa Fluor 488–conjugated goat anti-rabbit IgG (Molecular dose of 660 mg kg–1 i.p. twice a day for 4 d to reduce brain levels of glutathione in wild-type mice32. NAC was administered as a single 150 mg kg–1 intra- Probes). Confocal photomicrographs were acquired using 10-nm optical thickness sections. peritoneal injection 4–6 h before brain harvest to elevate neuronal glutathione levels in EAAC1–/– mice12,39,49. In some mice, the NAC injection was accom- Brain morphology measurements. Ventricle area was measured on three panied by BSO (1320 mg kg–1), to prevent de novo glutathione formation.© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience consecutive 30-mm coronal slices taken at the level of the anterior commissure, Biochemical glutathione determinations were performed by the NADPH- and on an additional three slices taken 2.7 mm posterior to the anterior dependent glutathione reductase method, as described previously21. In situ commissure. Width of the CA1 cell layer in each mouse was averaged from evaluation of reactive thiols, of which glutathione is the dominant species, was measurements from three consecutive hematoxylin-eosin–stained slices. Width performed using a fluorescent maleimide derivative50. After incubations under of the corpus callosum was measured on three consecutive slices stained with the designated conditions, hippocampal slices were washed with aCSF, fixed in lipophilic dye fluoromyelin (Molecular Probes) and counterstained with 4% paraformaldehyde, and incubated overnight at 4 1C with 2.5 mM Alexa propidium iodide (PI). Slice images were imported to Photoshop software Fluor 488 C5 maleimide (Molecular Probes) in phosphate-buffered saline for the image analysis, and measurements from the three consecutive slices were containing 2% goat serum, 0.2% Triton X-100 and 0.1% bovine serum averaged for each data point ‘n’. albumin. The slices were photographed with a confocal microscope and the images quantified as described for PI fluorescence. Behavioral studies. A total of 40 mice were studied, in two groups of 20. The first group consisted of 10 wild-type mice and 10 EAAC1–/– mice of age 11–12 Statistics. Data are expressed as means ± s.e.m. The behavioral studies were months. The second group consisted of 10 wild-type mice and 10 EAAC1–/– assessed using repeated-measures analysis of variance (ANOVA). The brain mice of age 6–8 weeks. To minimize the effects of social influences on behavior, morphology and biochemical measurements were assessed by ANOVA and the mice were housed individually in the testing room beginning 3 d before testing. Bonferroni test for multiple group comparisons. The fluorescent indicators As a first step, spontaneous open field activity was assessed to establish any used for cell death, immunostaining and thiol determinations were evaluated locomotor differences. On each of three consecutive days, open field activity with the Kruskal-Wallis test followed by Dunn’s test for multiple comparisons was recorded for 10 min after an initial 1-min adaptation period. Subsequently, between the wild-type and EAAC1–/– preparations. spatial learning and memory were evaluated by the Morris water maze task, as Note: Supplementary information is available on the Nature Neuroscience website. previously detailed46. The mice were trained first to locate a visible platform (days 1 and 2), and then to locate a hidden platform (days 3 through 5). The ACKNOWLEDGMENTS mice received two training sessions per day, each consisting of three 1-min trials We thank D. Burns for technical assistance and M. Yenari and S. Massa for with a 10-min intertrial interval. Swim speed and time to reach the platform critical reading of the manuscript. This work was supported by grants from (latency) was recorded with an EthoVision video tracking system (Noldus the US National Institutes of Health and the Department of Veterans Affairs. Information Technology). Mice that did not reach the platform within 60 s COMPETING INTERESTS STATEMENT were manually placed on the platform and assigned a latency interval of 60 s. The authors declare that they have no competing financial interests. Hippocampal slice preparation. Brains from wild-type and EAAC1–/– mice, Published online at http://www.nature.com/natureneuroscience/ 6–8 weeks of age, were vibratome-sectioned into 300-mm coronal slices. The Reprints and permissions information is available online at http://npg.nature.com/ slices were placed in ice-cold artificial cerebrospinal fluid (aCSF) containing reprintsandpermissions/ 130 mM NaCl, 3.5 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2, 20 mM NaHCO3 and 10 mM glucose at pH 7.2 while equilibrated with 95% 1. Danbolt, N.C. Glutamate uptake. Prog. Neurobiol. 65, 1–105 (2001). oxygen and 5% CO2. The aCSF osmolality was 290 ± 15 mOsm kg–1 as 2. Rothstein, J.D. et al. Localization of neuronal and glial glutamate transporters. Neuron 13, 713–725 (1994). determined by Wescor vapor pressure osmometer. All studies were done using 3. Shashidharan, P. et al. Immunohistochemical localization of the neuron-specific gluta- brain slices from wild-type and EAAC1–/– mice treated in parallel. Experiments mate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel were initiated by transferring the slices to a circulating bath of aCSF at 30 1C monoclonal antibody. Brain Res. 773, 139–148 (1997). that was continuously bubbled with 95% oxygen and 5% CO2. Glutamate and 4. Arriza, J.L., Eliasof, S., Kavanaugh, M.P. & Amara, S.G. Excitatory amino acid trans- porter 5, a retinal glutamate transporter coupled to a chloride conductance. Proc. Natl. oxidants were added for 30-min incubation intervals. Slices were then trans- Acad. Sci. USA 94, 4155–4160 (1997). ferred to fresh aCSF and maintained at 22 1C until harvested for cell death or 5. Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate immunostaining 3.5 h later. Where designated, 20 mM (+)-bicuculline (Tocris) transporter GLT-1. Science 276, 1699–1702 (1997). was incubated with the slices beginning 30 min before the H2O2 or SIN-1 6. Peghini, P., Janzen, J. & Stoffel, W. Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegenera- exposures to block GABAA receptor function36,47. tion. EMBO J. 16, 3822–3832 (1997). 7. Rothstein, J.D. et al. Knockout of glutamate transporters reveals a major role for Cell death determinations. Cell death in the brain slices was assessed as astroglial transport in excitotoxicity and clearance of glutamate. Neuron 16, 675–686 described previously48, with minor changes. Slices were incubated for 3 min (1996). with 5 mM PI, washed, fixed in 4% paraformaldehyde and stored in the dark at 8. Coco, S. et al. Non-synaptic localization of the glutamate transporter EAAC1 in cultured hippocampal neurons. Eur. J. Neurosci. 9, 1902–1910 (1997). 4 1C. Digitized images were prepared with a confocal fluorescent microscope 9. Sepkuty, J.P. et al. A neuronal glutamate transporter contributes to neurotransmitter within 24 h of staining. The PI signal intensity was measured in the CA1 cell GABA synthesis and epilepsy. J. Neurosci. 22, 6372–6379 (2002). body layer of each slice using a ‘region of interest’ mask of constant size. The 10. Zerangue, N. & Kavanaugh, M.P. Interaction of L-cysteine with a human excitatory amino background signal was measured from the stratum radiatum of each slice, acid transporter. J. Physiol. (Lond.) 493, 419–423 (1996). 11. Bendahan, A., Armon, A., Madani, N., Kavanaugh, M.P. & Kanner, B.I. Arginine 447 which contained no PI fluorescence, and subtracted from the value obtained in plays a pivotal role in substrate interactions in a neuronal glutamate transporter. J. Biol. the CA1 region. Values from three slices per brain were averaged for each ‘n’. Chem. 275, 37436–37442 (2000). 12. Wu, G., Fang, Y.Z., Yang, S., Lupton, J.R. & Turner, N.D. Glutathione metabolism and its Measurement of reactive oxygen species in hippocampal slices. Hippocampal implications for health. J. Nutr. 134, 489–492 (2004). slices were pre-incubated in 10 mM 5-(and 6-)carboxy-2¢,7¢-dichlorodihydro- 13. Dringen, R., Pfeiffer, B. & Hamprecht, B. Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. fluorescein diacetate (DCF; Molecular Probes) for 30 min to allow intracellular J. Neurosci. 19, 562–569 (1999). loading. After washing with aCSF, slices were exposed to H2O2 or SIN-1 for 14. Shanker, G., Allen, J.W., Mutkus, L.A. & Aschner, M. The uptake of cysteine in cultured 30 min. The slices were immediately photographed to quantify DCF fluores- primary astrocytes and neurons. Brain Res. 902, 156–163 (2001). NATURE NEUROSCIENCE VOLUME 9 [ NUMBER 1 [ JANUARY 2006 125
    • ARTICLES 15. Murphy, T.H., Schnaar, R.L. & Coyle, J.T. Immature cortical neurons are uniquely 34. Avshalumov, M.V. & Rice, M.E. NMDA receptor activation mediates hydrogen peroxide- sensitive to glutamate toxicity by inhibition of cystine uptake. FASEB J. 4, 1624–1633 induced pathophysiology in rat hippocampal slices. J. Neurophysiol. 87, 2896–2903 (1990). (2002). 16. Sagara, J.I., Miura, K. & Bannai, S. Maintenance of neuronal glutathione by glial cells. 35. Nowak, L., Bregestovski, P., Ascher, P., Herbet, A. & Prochiantz, A. Magnesium gates J. Neurochem. 61, 1672–1676 (1993). glutamate-activated channels in mouse central neurones. Nature 307, 462–465 17. Sato, H. et al. Distribution of cystine/glutamate exchange transporter, system x(c)-, in (1984). the mouse brain. J. Neurosci. 22, 8028–8033 (2002). 36. Grover, L.M. & Yan, C. Blockade of GABAA receptors facilitates induction of 18. Baker, D.A. et al. Neuroadaptations in cystine-glutamate exchange underlie cocaine NMDA receptor-independent long-term potentiation. J. Neurophysiol. 81, 2814– relapse. Nat. Neurosci. 6, 743–749 (2003). 2822 (1999). 19. Wang, X.F. & Cynader, M.S. Astrocytes provide cysteine to neurons by releasing 37. Mazor, D. et al. Red blood cell permeability to thiol compounds following oxidative glutathione. J. Neurochem. 74, 1434–1442 (2000). stress. Eur. J. Haematol. 57, 241–246 (1996). 20. Dringen, R., Gutterer, J.M., Gros, C. & Hirrlinger, J. Aminopeptidase N mediates the 38. Parsons, J.L. & Chipman, J.K. The role of glutathione in DNA damage by potassium© 2006 Nature Publishing Group http://www.nature.com/natureneuroscience utilization of the glutathione precursor CysGly by cultured neurons. J. Neurosci. Res. 66, bromate in vitro. Mutagenesis 15, 311–316 (2000). 1003–1008 (2001). 39. Corcoran, G.B. & Wong, B.K. Role of glutathione in prevention of acetaminophen- 21. Chen, Y. & Swanson, R.A. The glutamate transporters EAAT2 and EAAT3 mediate induced hepatotoxicity by N-acetyl-L-cysteine in vivo: studies with N-acetyl-D-cysteine cysteine uptake in cortical neuron cultures. J. Neurochem. 84, 1332–1339 (2003). in mice. J. Pharmacol. Exp. Ther. 238, 54–61 (1986). 22. Himi, T., Ikeda, M., Yasuhara, T., Nishida, M. & Morita, I. Role of neuronal glutamate 40. Umansky, V. et al. Glutathione is a factor of resistance of Jurkat leukemia cells to nitric transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical oxide-mediated apoptosis. J. Cell. Biochem. 78, 578–587 (2000). neurons. J. Neural Transm. 110, 1337–1348 (2003). 41. Zerangue, N. & Kavanaugh, M.P. Flux coupling in a neuronal glutamate transporter. 23. Dringen, R. Metabolism and functions of glutathione in brain. Prog. Neurobiol. 62, Nature 383, 634–637 (1996). 649–671 (2000). 42. Smith, C.P. et al. Assignment of the gene coding for the human high-affinity glutamate 24. Jain, A., Martensson, J., Stole, E., Auld, P.A. & Meister, A. Glutathione deficiency leads transporter EAAC1 to 9p24: potential role in dicarboxylic aminoaciduria and neuro- to mitochondrial damage in brain. Proc. Natl. Acad. Sci. USA 88, 1913–1917 (1991). degenerative disorders. Genomics 20, 335–336 (1994). 25. Schulz, J.B., Lindenau, J., Seyfried, J. & Dichgans, J. Glutathione, oxidative stress and 43. Gonzalez, M.I., Kazanietz, M.G. & Robinson, M.B. Regulation of the neuronal glutamate neurodegeneration. Eur. J. Biochem. 267, 4904–4911 (2000). transporter excitatory amino acid carrier-1 (EAAC1) by different protein kinase C 26. Yang, Y., Kinney, G.A., Spain, W.J., Breitner, J.C. & Cook, D.G. Presenilin-1 and subtypes. Mol. Pharmacol. 62, 901–910 (2002). intracellular calcium stores regulate neuronal glutamate uptake. J. Neurochem. 88, 44. Canolle, B. et al. Glial soluble factors regulate the activity and expression of the neuronal 1361–1372 (2004). glutamate transporter EAAC1: implication of cholesterol. J. Neurochem. 88, 27. Morris, R. Development of a water-maze procedure for studying spatial learning in the 1521–1532 (2004). rat. J. Neurosci. Methods 11, 47–60 (1984). 45. Aoyama, K. et al. Acidosis causes endoplasmic reticulum stress and caspase-12- 28. Hogg, N., Darley-Usmar, V.M., Wilson, M.T. & Moncada, S. Production of hydroxyl mediated astrocyte death. J. Cereb. Blood Flow Metab. 25, 258–370 (2005). radicals from the simultaneous generation of superoxide and nitric oxide. Biochem. 46. Suh, S.W. et al. Hypoglycemic neuronal death and cognitive impairment are prevented J. 281, 419–424 (1992). by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia. J. Neu- 29. Himi, T., Ikeda, M., Yasuhara, T. & Murota, S.I. Oxidative neuronal death caused by rosci. 23, 10681–10690 (2003). glutamate uptake inhibition in cultured hippocampal neurons. J. Neurosci. Res. 71, 47. Hochman, D.W. & Schwartzkroin, P.A. Chloride-cotransport blockade desynchronizes 679–688 (2003). neuronal discharge in the ‘‘epileptic’’ hippocampal slice. J. Neurophysiol. 83, 406–417 30. Hogg, N., Singh, R.J. & Kalyanaraman, B. The role of glutathione in the transport and (2000). catabolism of nitric oxide. FEBS Lett. 382, 223–228 (1996). 48. Yin, H.Z., Sensi, S.L., Ogoshi, F. & Weiss, J.H. Blockade of Ca2+-permeable 31. Beckman, J.S. & Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: the good, AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn2+ accumula- the bad, and ugly. Am. J. Physiol. 271, C1424–C1437 (1996). tion and neuronal loss in hippocampal pyramidal neurons. J. Neurosci. 22, 1273–1279 32. Griffith, O.W. & Meister, A. Potent and specific inhibition of glutathione synthesis by (2002). buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J. Biol. Chem. 254, 49. Meister, A., Anderson, M.E. & Hwang, O. Intracellular cysteine and glutathione delivery 7558–7560 (1979). systems. J. Am. Coll. Nutr. 5, 137–151 (1986). 33. Crow, J.P. Dichlorodihydrofluorescein and dihydrorhodamine 123 are sensitive indica- 50. Lantz, R.C., Lemus, R., Lange, R.W. & Karol, M.H. Rapid reduction of intracellular tors of peroxynitrite in vitro: implications for intracellular measurement of reactive glutathione in human bronchial epithelial cells exposed to occupational levels of toluene nitrogen and oxygen species. Nitric Oxide 1, 145–157 (1997). diisocyanate. Toxicol. Sci. 60, 348–355 (2001). 126 VOLUME 9 [ NUMBER 1 [ JANUARY 2006 NATURE NEUROSCIENCE