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. 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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
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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 email@example.com 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 firstname.lastname@example.org 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 email@example.com 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
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. 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