Effect of Nitrous Oxide Use on Long-term Neurologic and Neuropsychological Outcome in Patients Who Received Temporary Proximal Artery Occlusion during Cerebral Aneurysm Clipping Surgery

Our study was based exclusively on a post hoc  analysis of the IHAST database. IHAST was a large (1,001-patient), international, multicenter, randomized, partially blinded, prospective, clinical trial. Details regarding trial design are described elsewhere.1In brief, nonpregnant adults with a preoperative World Federation of Neurological Surgeons Score of I, II, or III and who had aneurysmal subarachnoid hemorrhage no more than 14 days before surgery were eligible for enrollment. Specific exclusion criteria included a body mass index 35 kg/m²or greater, any cold-related disorder (e.g. , Raynaud disease), and tracheal intubation at the time of enrollment. Extensive information regarding the patients' pre–subarachnoid hemorrhage health status and events occurring between the time of hospital admission and surgery were collected. The study was approved by each center's local Human Studies Committee, and informed consent was obtained from each patient or their legal representative. All study personnel, except the anesthesiologist involved in each patient's intraoperative care, were blinded to treatment assignment.

Anesthesia was limited to either intravenous thiopental or etomidate for induction of anesthesia, and inhaled isoflurane or desflurane for maintenance, supplemented by either intravenous fentanyl or remifentanil. Inhaled nitrous oxide use was at the discretion of the anesthesiologists, and no limitations were imposed by the study protocol on the concentration of nitrous oxide administered.

After the induction of anesthesia, an esophageal temperature probe was inserted, and the patient was positioned for surgery. In patients randomly assigned to hypothermia, esophageal temperature was reduced as quickly as possible, with the goal of achieving a temperature between 32.5° and 33.5°C by the time a clip was applied to the first aneurysm. Temperature in patients randomly assigned to normothermia was kept between 36° and 37°C. Rewarming of hypothermic patients began after the last aneurysm had been secured and was continued until normothermia was achieved. Use of proximal temporary artery occlusion to facilitate clipping of the aneurysm or administration of a supplemental cerebral metabolism suppressant anesthetic (etomidate or thiopental) intraoperatively was at the discretion of the individual surgeons and anesthesiologists.

No attempt was made to control postoperative care, but all adverse events, procedures, and other aspects of treatment were monitored for either 14 days or until hospital discharge (if this occurred before 14 days). Of particular note, a clinical diagnosis of DIND was made if there was (1) a decrease in the Glasgow Coma Score with alteration in level of consciousness or (2) development of a new or worsening focal neurologic deficit after the exclusion of other causes (e.g. , drug effect, hydrocephalus, aneurysmal rebleeding, intracranial hematoma, cerebral edema, or metabolic disturbances such as hypoxia, hyponatremia, or aberrant glucose homeostasis).

A final follow-up examination was conducted approximately 3 months after surgery. Outcome measures included (1) the modified Glasgow Outcome Score (GOS; this was the primary outcome measure for the trial)15; (2) the Rankin Disability Score16; (3) the Barthel Activities of Daily Living Index17; (4) the National Institutes of Health Stroke Scale (NIHSS)18; (5) the site to which the patient was discharged from the hospital where surgery was performed (e.g. , to home, an acute care hospital, or a chronic care/rehabilitation facility); and (6) a neuropsychological battery that included the Benton Visual Retention Test,19Controlled Oral Word Association,20Rey-Osterrieth Complex Figure Test,21Grooved Pegboard, and Trail Making Tests.22Details regarding neuropsychological testing and scoring can be found elsewhere.23T scores for individual tests (after adjustment for age and education) were averaged to obtain a single composite score if at least three neuropsychological tests were completed; a composite score of 30 or less (2 SDs below the population norm of 50) was considered evidence of neuropsychological impairment. We also determined the number of subjects who were impaired (T score < 30) on at least one test in the battery, regardless of the composite score.

For living patients who were unable to complete the neuropsychological tests because of their overall gross neurologic status, imputed impairment was determined in a manner described elsewhere for the entire IHAST population.23Briefly, a computerized imputation process was developed which determined the likelihood of neuropsychological impairment based on scores obtained from four tests of gross neurologic function (i.e. , GOS, Rankin Score, NIHSS Score, and Barthel Index of Daily Living) derived from data obtained from 873 patients in the original IHAST trial who were able to complete neuropsychological testing.1The imputation process was based on relations between performance on tests of gross function and neuropsychological function in other patients enrolled in the IHAST trial. This process was only used to impute the likelihood of impairment on the composite score in living patients if the composite score was not directly computed using the patient's actual scores. The category “impairment of 1 or more neuropsychological tests” was imputed only if no neuropsychological test was completed and there was imputed impairment of the composite score. All evaluations were performed by trained examiners who were certified by the University of Iowa Steering Committee, Iowa City, Iowa.

All data analysis was conducted by the Data Management Center at the University of Iowa using SAS version 9.1.3 (SAS Institute, Inc., Cary, NC). Univariate comparisons of various measures in patients who did or did not receive nitrous oxide were performed using a Student t  test, Pearson chi-square test, or Fisher exact test depending on the characteristics and distribution of the data. It was not possible to structure the analysis according to nitrous oxide dose because nitrous oxide use was reported in the IHAST database as either used or not used.

All neurologic and neuropsychological outcomes were analyzed using both univariate and multivariate logistic regression. For binary outcomes, standard logistic regression analyses were performed and, for ordered categorical outcomes with more than two categories, cumulative logistic (proportional odds) models were used. Because the use of nitrous oxide was not based on random assignment, multivariate analyses were performed to assess the effect of nitrous oxide on outcomes after adjusting for a standard set of covariates, determined by IHAST Coordinating Center to be important covariates to include in all post hoc  analyses of neurologic and neuropsychological outcomes of the IHAST trial. The covariates for the multivariable analysis included race (white vs.  nonwhite), age, sex, baseline World Federations of Neurological Surgeons Score, baseline NIHSS score, Fisher grade, history of hypertension, time from subarachnoid hemorrhage to surgery, largest aneurysm size (1–11, 12–24, ≥ 25 mm in greatest dimension), aneurysm location (posterior vs.  anterior), and IHAST treatment assignment (normothermic vs.  hypothermic). For analysis purposes, duration of stay in the intensive care unit, total duration of hospitalization, and destination after discharge from the treating institution were stratified as binary responses (< 5 days vs. > 5 days, < 15 days vs. > 15 days, and home vs.  other facility or death, respectively). GOS was treated as an ordered categorical variable using all possible responses (1 = minor or no disability, 2 = moderate disability, 3 = severe disability, 4 = vegetative state, 5 = death) and also using a binary response (1 vs.  others). DIND was treated as a binary response (yes vs.  no), NIHSS was analyzed using five ordered categories (0 = no deficit, 1–7 = mild deficit, 8–14 = moderate deficit, 15–42 = severe deficit, death), Rankin score was treated as a binary variable (0–1 = minimal or no deficit, > 1 = significant deficit), and Barthel Activities of Daily Living Index score was treated as a binary variable (95–100 = minimal to no impairment, < 95 = impairment).1Specific details related to the scoring of neuropsychological tests can be found elsewhere.23Briefly, the results of each test were compared with normative data (adjusted for age, sex, and years of education), with a binary outcome (presence or absence of impairment) determined for each test. For the current report, two binary neuropsychological outcomes are included: impairment for the composite score and impairment on any individual test.

Because the IHAST study was a randomized trial evaluating whether intraoperative hypothermia would improve neurologic outcomes, initial analyses were performed to evaluate whether the effect of the randomized treatment (normothermic vs.  hypothermic) differed for patients who received nitrous oxide versus  those who did not. These analyses were performed using models that included nitrous oxide use (no vs.  yes), IHAST treatment assignment (normothermic vs.  hypothermic), and the nitrous oxide–by–treatment assignment interaction effect. After confirming that there were no significant interaction effects, subsequent logistic regression models that included nitrous oxide use as the only explanatory variable were used to assess the univariate association of nitrous oxide use on outcomes. Because the explanatory variable of interest for this investigation was nitrous oxide use, the findings from the multiple logistic regression models are summarized by presenting the odds ratio (OR) and corresponding 95% confidence interval (CI) for nitrous oxide use. For all logistic regression analyses, the models are parameterized so that an OR significantly greater than 1.0 would indicate an increased likelihood of a worse outcome in patients receiving nitrous oxide. In all cases, two-sided tests were performed with P ≤ 0.05 used to denote statistical significance.

Details regarding the primary IHAST trial results can be found elsewhere.1,23Briefly, induced hypothermia had no effect on any gross neurologic or neuropsychological variable studied.

In the subset of 441 IHAST patients who underwent intraoperative cerebral artery occlusion to facilitate aneurysm clipping, demographics and data pertaining to preoperative neurologic status, stratified based on intraoperative nitrous oxide use, can be found in table 1. Groups did not differ with regard to sex, preoperative medical history, or time from initial subarachnoid hemorrhage to induction of anesthesia. There were statistically significant, but probably clinically inconsequential, differences between groups with respect to age (50 ± 11 vs.  52 ± 12 yr for nitrous oxide and no nitrous oxide groups, respectively; P = 0.041) and fraction of patients who were white, not of Hispanic origin (76% vs.  85% for nitrous oxide and no nitrous oxide groups, respectively; P = 0.019). Preoperative World Federation of Neurological Surgeons score data were equivalent. There was a significant difference between groups with respect to both initial NIHSS score (P = 0.035) and Fisher grade (P = 0.01).

Aneurysm characteristics and intraoperative data were largely equivalent between the two groups and are summarized in table 2. There was no difference in the fraction of patients in each group who received either isoflurane or desflurane during maintenance of general anesthesia. It should be noted that the fraction of patients who received isoflurane and desflurane in each group was greater than the total number of patients in that group, reflecting that in a few patients, the volatile agent was changed during the course of the same general anesthetic.

Patients in the nitrous oxide group had a significantly greater mean arterial blood pressure (83 ± 14 vs.  77 ± 14 mmHg for no nitrous oxide; P < 0.001) and blood glucose (141 ± 35 vs.  126 ± 35 mg/dl for no nitrous oxide; P < 0.001) at the time of first permanent clip placement, as well as a greater time interval between induction of anesthesia and placement of the first permanent clip (240 ± 80 vs.  211 ± 75 min for no nitrous oxide; P < 0.001) and the time between placement of the last permanent clip and arrival in the recovery area (116 ± 32 vs.  94 ± 34 min for no nitrous oxide; P < 0.001). A greater fraction of patients in the nitrous oxide group also received a supplemental metabolic depressant (i.e. , protective) anesthetic agent before vessel occlusion and aneurysm clipping (46% vs.  35% for no nitrous oxide; P = 0.024). Although there was no difference in the fraction of patients who had a temporary clip placed for longer than 20 min, the mean duration of iatrogenic temporary arterial occlusion was greater in the nitrous oxide group (11.9 ± 13.1 vs.  9.3 ± 7.7 min for no nitrous oxide; P = 0.012). Statistically significant but probably clinically inconsequential differences existed between groups in core temperature both at the time of arrival in the operating suite and 2 h after surgery, but not at the time of first permanent clip placement. Although a lower fraction of nitrous oxide–treated patients had an aneurysm that was judged difficult or very difficult to expose (39% vs.  51% for no nitrous oxide; P = 0.008), there was no difference between groups in the fraction of patients judged to have moderate or severe brain swelling on dural opening. Other intraoperative factors were equivalent.

Postoperative data are summarized in table 3. After both univariate and multivariate analysis (which corrected for factors thought to influence outcome), a significantly greater fraction of patients in the nitrous oxide group had an intensive care unit duration of stay of 5 or more days compared with the group that did not receive nitrous oxide (69% and 46% for nitrous oxide and no nitrous oxide groups, respectively; univariate and multivariate P < 0.001 for both). There was no difference between groups in the fraction of patients with an overall hospital duration of stay of 15 or more days (59% vs.  55% for nitrous oxide and no nitrous oxide groups, respectively; P = 0.424 and P = 0.209 for univariate and multivariate analysis, respectively). Further, a greater fraction of patients in the group that did not receive nitrous oxide were not discharged to home (48% vs.  33% for the nitrous oxide group; P = 0.001 and P = 0.006 for univariate and multivariate analyses, respectively).

Both early (i.e. , DIND) and late (i.e. , 3-month postoperative assessments of neurologic and neuropsychological) outcome results are summarized in table 4. In those who received nitrous oxide, there was a greater fraction who displayed postoperative neurologic changes consistent with DIND (28% vs.  21% in the no nitrous oxide group) based on multivariate (adjusted OR, 1.78; CI, 1.08–2.95; P = 0.025) but not univariate (P = 0.108) analysis.

There was no significant association between nitrous oxide use and outcome at 3 months after subarachnoid hemorrhage as measured by GOS stratified as a binary variable (univariate P = 0.059; adjusted OR, 0.70; CI, 0.45–1.10; multivariate P = 0.123). When stratified as an ordered categorical variable, use of nitrous oxide was associated with improved GOS score on univariate (unadjusted OR, 0.67; CI, 0.46–0.99; P = 0.043) but not multivariate logistic regression analysis (adjusted OR, 0.67; CI, 0.44–1.03; P = 0.065). There was no association between nitrous oxide use and outcome based on the Rankin Disability Score (univariate P = 0.078; adjusted OR, 0.74; CI, 0.47–1.16; multivariate P = 0.192), NIHSS score (univariate P = 0.741; adjusted OR, 1.02; CI, 0.66–1.56; multivariate P = 0.937), or Barthel Index of Daily Living (univariate P = 0.076; adjusted OR, 0.69; CI, 0.38–1.25; multivariate P = 0.220).

Regarding the evaluation of neuropsychological outcome 3 months after subarachnoid hemorrhage, impairment of the composite score was imputed for 10 patients (5.0%) in the nitrous oxide group and 11 patients (4.5%) in the no nitrous oxide group (P = 0.81). Impairment on more than one neuropsychological test was imputed for 8 patients (4.0%) in the nitrous oxide group and 8 patients (3.3%) in the no nitrous oxide group (P = 0.69). There was no difference in the fraction of patients who exhibited impairment of the neuropsychological composite score (20% for nitrous oxide group and 20% in the no nitrous oxide group; univariate P = 0.918; adjusted OR, 0.81; CI, 0.44–1.49; multivariate P = 0.493). However, a lesser fraction of patients in the group that received nitrous oxide (54%) exhibited impairment on at least one test of neuropsychological function compared with the group that did not receive nitrous oxide (67%) (univariate P = 0.008; adjusted OR, 0.56; CI, 0.36–0.89; multivariate P = 0.013).

In this post hoc  investigation of 441 patients having cerebral aneurysm surgery, in whom temporary occlusion of a cerebral artery was used, intraoperative use of nitrous oxide had no detrimental effect on neurologic status, functional status, or neuropsychological function 3 months after surgery. Nitrous oxide use was associated with both an increased odds for the development of DIND and fewer patients being discharged from the intensive care unit within 5 days. However, despite no difference between groups in the fraction of patients requiring an overall hospital stay of 15 or more days, a greater fraction of those who received nitrous oxide were discharged from their treating hospital to home versus  other acute care hospitals or chronic/rehabilitative facilities.

Some previous investigations have reported that nitrous oxide adversely effects the brain because of its effect on cerebral metabolism or intracranial pressure.24–28Other investigations using animal models have reported that nitrous oxide can augment injury in the ischemic brain.6,7,29Nevertheless, these effects have not been validated in humans. Additional research using animal models of ischemia have reported that exposure to nitrous oxide, a known N -methyl-d-aspartate receptor antagonist,30,31can reduce infarct size probably by limiting injury due to glutamate excitotoxicity.5,30 

A previous investigation by our group that studied all 1,001 patients in the IHAST database determined that the use of nitrous oxide during cerebral aneurysm clipping had no effect on the development of DIND (a manifestation of cerebral vasospasm) or both long-term (i.e. , 3 months after surgery) gross neurologic and neuropsychological function.2However, a limitation of our previous investigation was that many of the 1,001 patients may not have experienced a meaningful (i.e. , outcome-determining) cerebral ischemic insult at the same time that nitrous oxide was being administered. For example, not all patients who have a subarachnoid hemorrhage and undergo surgical clipping have alterations of cerebral hemodynamics sufficient to produce clinically important focal or global cerebral ischemic insults.1,32Further, intraoperative nitrous oxide should have little or no impact on ischemic episodes that occurred before surgery (i.e. , at the time of initial hemorrhage) due to the temporal relation. In our current investigation, we analyzed data exclusively from patients in whom temporary occlusion of a major cerebral artery was used to facilitate placement of a permanent clip on the aneurysm. In this subgroup analysis, ischemic events to the brain were not only more likely than in the 1,001 IHAST patients as a whole, but more importantly, any intraoperative ischemic events would have occurred during  exposure to nitrous oxide in many patients.

Use of temporary proximal vascular occlusion to facilitate placement of a permanent aneurysm clip is a relatively common practice. In the IHAST investigation, 44% of 1,001 patients had intraoperative proximal vascular occlusion.1This technique has been reported to reduce the risk of aneurysm rupture and helps to facilitate surgical dissection, potentially improving the rate of successful placement of a permanent clip.33However, use of temporary proximal vessel occlusion has adverse consequences as well. In patients having aneurysm clipping with somatosensory evoked potential monitoring, Mizoi and Yoshimoto34discovered a loss of signals in 43% of patients at the time of temporary proximal vessel occlusion. In a similar investigation, Schick et al.  35reported that in patients in whom proximal vessel occlusion was used, complete loss of somatosensory evoked potential signals occurred in 38%. Further, given the time course of signal loss, the authors concluded that there was no safe permissible time for temporary arterial occlusion, a finding supported by other investigations.36,37Additional studies have reported that a longer duration of temporary occlusion is associated with an increased risk of infarction.38,39Given that all patients included in our analysis underwent selective arterial occlusion, important intraoperative ischemic events were likely in a large fraction of these 411 patients. Of note, there was no difference between groups in the fraction of patients who underwent occlusion of specific vessels (untabulated data).

Our investigation evaluated one metric of short-term neurologic function: DIND, the clinical manifestation of cerebral vasospasm. There are mechanistic reasons to expect that nitrous oxide could influence this event. In in vivo  studies, methylation of homocysteine, a reaction catalyzed by the enzyme methionine synthase, results in the production of methionine. Nitrous oxide is a known inhibitor of methionine synthase and exposure to nitrous oxide has been reported to acutely increase serum homocysteine concentrations.14Among its many effects, homocysteine is known to increase platelet production of thromboxane A², a potent vasoconstrictor.40In our previous investigation that included data from all 1,001 IHAST subjects, nitrous oxide use was not associated with the development of DIND (OR, 1.29; CI, 0.91–1.83; P = 0.157).2However, when only patients in whom temporary arterial occlusion was used were included in the analysis, use of nitrous oxide increased the odds of developing DIND postoperatively (OR, 1.78; CI, 1.08-2.95; P = 0.025). It is currently unknown whether use of temporary vessel occlusion served as an independent risk factor to increase the risk for development of DIND. This issue is currently being evaluated by another post hoc  probe of the IHAST database.

Multiple possible explanations may account for increased DIND in nitrous oxide–treated patients. First, it is possible that the use of a temporary arterial clip resulted in irritation of the vascular smooth muscle, and, after exposure to increased serum homocysteine concentrations, increased the risk for vasospasm. Another possible explanation is that nitrous oxide augmented the ischemic insult to neurons and glia during temporary arterial occlusion and this, in turn, accounted for the increased number of patients experiencing short-term neurologic deficits. This finding may, in part, account for the greater fraction of patients in the nitrous oxide group who were discharged from the intensive care unit in 5 or more days.

The development of vasospasm after subarachnoid hemorrhage, independent of nitrous oxide use, is reported to reduce the chance of a good recovery by a factor of 3.41However, the increased rate of DIND in the nitrous oxide group in our research did not influence overall hospital duration of stay, discharge destination, or any metric of long-term gross neurologic or neuropsychological function. It is possible that, although nitrous oxide increased the incidence of symptomatic DIND, the severity of DIND associated with nitrous oxide use was not sufficient to modulate long-term outcome.

The long-term outcomes in this “higher-risk” group of 441 patients is generally consistent with our previous analysis of all 1,001 IHAST patients2; specifically, use of nitrous oxide intraoperatively had no detrimental effect on long-term gross neurologic or neuropsychological function. Our analysis showed a significantly reduced risk of a poor outcome at 3 months based on the categorized GOS after univariate analysis; however, that effect was found to be insignificant after multivariate logistic regression analysis. Given that nitrous oxide use was not the randomized variable in this investigation, based on our data, we conclude that use of nitrous oxide had no significant effect on GOS 3 months after surgery. However, unlike our previous analysis, this current investigation, which included only patients who had temporary occlusion of a major cerebral artery, revealed a significantly reduced risk of impairment on one or more neuropsychological tests at 3 months in those who received nitrous oxide intraoperatively. We use caution when interpreting this finding. This apparent “protective” effect by nitrous oxide may be due to many factors. For example, the baseline neuropsychological function of patients included in this analysis is unknown. It is possible that there were differences in baseline cognitive function between groups. Also, differences in various intraoperative factors, such as a greater use of neuroprotective agents intraoperatively, a 6-mmHg greater mean arterial blood pressure at the time of clip placement, and less difficulty with aneurysm exposure in those who received nitrous oxide may have confounded our results (table 2). Variations in postoperative care, factors not recorded as part of the IHAST trial, also may have affected our results. Finally, this positive finding in favor of nitrous oxide use could have been the result of a type I statistical error.

There are several limitations of our study that deserve comment. First, because use of nitrous oxide was not a randomized variable, statistical correction with factors thought to influence outcome was necessary. To minimize bias, in the multivariate logistic regression analysis, we corrected for the standard set of variables, determined by the IHAST coordinating center, that are being applied to all post hoc  investigations using the IHAST database. This standard set of covariates was chosen either because, using the data from the parent investigation, an individual item was a prerandomization variable found to be univariately associated with outcome or because the extensive subarachnoid hemorrhage literature suggests its link with outcome.

Another shortcoming of this current investigation is the potential for type I statistical error. For every comparison performed using the same data, the chance of a false-positive result increases with each comparison. Given that this article describes a post hoc  analysis of a data set, one must consider that our significant findings in this investigation may describe false positives. However, given the few positive comparisons reported in our analysis, the risk for a family-wise error remains small.

There also is the potential for type II statistical error given that effective sample sizes were not determined as part of the study design. The sample size for the original IHAST analysis (target n = 1,000, with approximately 500 per treatment group) was selected to permit detection of a 10 percentage-point difference (e.g. , 60% vs.  70%) in GOS between groups with statistical power of 91% using a two-sided, α= 0.05 level test.1For the current study, the effective sample size (n = 441; 199 with nitrous oxide, 242 without nitrous oxide) provides statistical power of 82% to detect a 13 percentage-point difference (e.g. , 60% vs.  73%) between groups, but only provides statistical power of 60% to detect a 10 percentage-point difference between groups. However, given the numerous instances in which the data tended to show an improvement—not harm—associated with nitrous oxide use, it seems reasonable to conclude that at least no profound harmful long-term effect was inflicted on patients with the use of nitrous oxide in conjunction with temporary arterial occlusion during aneurysm surgery.

Finally, another limitation of this investigation was the lack of measurement of cerebral blood flow or monitoring for evidence of cerebral ischemia during cerebral arterial occlusion. Use of such monitoring modalities was not required by the original IHAST investigation and, if performed, these data were not recorded in the IHAST database. Without these data, it is unknown whether differences existed between groups with respect to both the number of patients who experienced ischemic episodes and the extent of ischemia. We attempted to estimate the severity of any ischemic insults by assessing other variables within the IHAST database (e.g. , duration of temporary occlusion, mean arterial blood pressure, use of neuroprotective agents).

In summary, use of nitrous oxide in a group of patients at high risk for cerebral ischemia had no detrimental effect on long-term gross neurologic or neuropsychological function. Nitrous oxide use was associated with an increased risk of developing DIND, but this did not correlate with long-term outcome. Given the findings of this investigation, we confirm our previous impression from the study of nitrous oxide in the entire IHAST population: There is no evidence to support the unconditional avoidance of nitrous oxide in patients at risk for cerebral ischemia.

The authors thank Ming Yang, B.S., and Emine Baym, B.S. (Graduate Research Assistants, Department of Biostatistics, University of Iowa School of Public Health, Iowa City, Iowa), for their assistance with data compilation and statistical calculations. The authors also thank Julie Weeks, M.P.T. (Program Associate, Department of Anesthesiology, University of Iowa Carver College of Medicine, Iowa City, Iowa), for her help in facilitating data organization.

The members of IHAST were as follows: University of Iowa—Steering Committee:  M. Todd, B. Hindman, W. Clarke, K. Chaloner, J. Torner, P. Davis, M. Howard, D. Tranel, S. Anderson; Clinical Coordinating Center:  M. Todd, B. Hindman, J. Weeks, L. Moss, J. Winn; Data Management Center:  W. Clarke, K. Chaloner, M. Wichman, R. Peters, M. Hansen, D. Anderson, J. Lang, B. Yoo; Physician Safety Monitor:  H. Adams; Project Advisory Committee:  G. Clifton (University of Texas, Houston, Texas), A. Gelb (University of California, San Francisco, California), C. Loftus (Temple University, Philadelphia, Pennsylvania), A. Schubert (Cleveland Clinic, Cleveland, Ohio); Physician Protocol Monitor:  D. Warner (Duke University, Durham, North Carolina); Data and Safety Monitoring Board:  W. Young, Chair (University of California, San Francisco, California), R. Frankowski (University of Texas Health Science Center at Houston School of Public Health, Houston, Texas), K. Kieburtz (University of Rochester School of Medicine and Dentistry, Rochester, New York), D. Prough, University of Texas Medical Branch, Galveston, Texas), L. Sternau (Mt. Sinai Medical Center, Miami, Florida); National Institutes of Health, National Institute of Neurologic Disease and Stroke, Bethesda, Maryland:  J. Marler, C. Moy, B. Radziszewska.

Participating centers (the number of randomized patients at each center is listed in parentheses): Addenbrooke's Hospital, Cambridge, United Kingdom (93):  B. Matta, P. Kirkpatrick, D. Chatfield, C. Skilbeck, R. Kirollos, F. Rasulo, K. English, C. Duffy, K. Pedersen, N. Scurrah, R. Burnstein, A. Prabhu, C. Salmond, A. Blackwell, J. Birrell, S. Jackson; University of Virginia Health System, Charlottesville, Virginia (86):  N. Kassell, T. Pajewski, H. Fraley, A. Morris, T. Alden, M. Shaffrey, D. Bogdonoff, M. Durieux, Z. Zuo, K. Littlewood, E. Nemergut, R. Bedford, D. Stone, P. Balestrieri, J. Mason, G. Henry, P. Ting, J. Shafer, T. Blount, L. Kim, A. James, E. Farace, L. Clark, M. Irons, T. Sasaki, K. Webb; Auckland City Hospital, Auckland, New Zealand (69):  T. Short, E. Mee, J. Ormrod, J. Jane, T. Alden, P. Heppner, S. Olson, D. Ellegala, C. Lind, J. Sheehan, M. Woodfield, A. Law, M. Harrison, P. Davies, D. Campbell, N. Robertson, R. Fry, D. Sage, S. Laurent, C. Bradfield, K. Pedersen, K. Smith, Y. Young, C. Chambers, B. Hodkinson, J. Biddulph, L. Jensen, J. Ogden, Z. Thayer, F. Lee, S. Crump, J. Quaedackers, A. Wray, V. Roelfsema; Sozialmedizinisches Zentrum Ost–Donauspital, Vienna, Switzerland (58):  R. Greif, G. Kleinpeter, C. Lothaller, E. Knosp, W. Pfisterer, R. Schatzer, C. Salem, W. Kutalek, E. Tuerkkan, L. Koller, T. Weber, A. Buchmann, C. Merhaut, M. Graf, B. Rapf; Harborview Medical Center, Seattle, Washington (58):  A. Lam, D. Newell, P. Tanzi, L. Lee, K. Domino, M. Vavilala, J. Bramhall, M. Souter, G. Britz, H. Winn, H. Bybee; St. Vincent's Public Hospital, Melbourne, Australia (57):  T. Costello, M. Murphy, K. Harris, C. Thien, D. Nye, T. Han, P. McNeill, B. O'Brien, J. Cormack, A. Wyss, R. Grauer, R. Popovic, S. Jones, R. Deam, G. Heard, R. Watson, L. Evered, F. Bardenhagen, C. Meade, J. Haartsen, J. Kruger, M. Wilson; University of Iowa Health Care, Iowa City, Iowa (56):  M. Maktabi, V. Traynelis, A. McAllister, P. Leonard, B. Hindman, J. Brian, F. Mensink, R. From, D. Papworth, P. Schmid, D. Dehring, M. Howard, P. Hitchon, J. VanGilder, J. Weeks, L. Moss, K. Manzel, S. Anderson, R. Tack, D. Taggard, P. Lennarson, M. Menhusen; University of Western Ontario, London, Ontario, Canada (53):  A. Gelb, S. Lownie, R. Craen, T. Novick, G. Ferguson, N. Duggal, J. Findlay, W. Ng, D. Cowie, N. Badner, I. Herrick, H. Smith, G. Heard, R. Peterson, J. Howell, L. Lindsey, L. Carriere, M. von Lewinski, B. Schaefer, D. Bisnaire, P. Doyle-Pettypiece, M. McTaggart; Keck School of Medicine at University of Southern California, Los Angeles, California (51):  S. Giannotta, V. Zelman, E. Thomson, E. Babayan, C. McCleary, D. Fishback; University of Michigan Medical Center, Ann Arbor, Michigan (41):  S. Samra, B. Thompson, W. Chandler, J. Mcgillicuddy, K. Tremper, C. Turner, P. Smythe, E. Dy, S. Pai, V. Portman, J. Palmisano, D. Auer, M. Quigley, B. Giordani, A. Freymuth, P. Scott, R. Silbergleit, S. Hickenbottom; University of California San Francisco, San Francisco, California (39):  L. Litt, M. Lawton, L. Hannegan, D. Gupta, P. Bickler, B. Dodson, P. Talke, I. Rampil, B. Chen, P. Wright, J. Mitchell, S. Ryan, J. Walker, N. Quinnine, C. Applebury; Alfred Hospital, Melbourne, Australia (35):  P. Myles, J. Rosenfeld, J. Hunt, S. Wallace, P. D'Urso, C. Thien, J. McMahon, S. Wadanamby, K. Siu, G. Malham, J. Laidlaw, S. Salerno, S. Alatakis, H. Madder, S. Cairo, A. Konstantatos, J. Smart, D. Lindholm, D. Bain, H. Machlin, J. Moloney, M. Buckland, A. Silvers, G. Downey, A. Molnar, M. Langley, D. McIlroy, D. Daly, P. Bennett, L. Forlano, R. Testa, W. Burnett, F. Johnson, M. Angliss, H. Fletcher; Toronto Western Hospital, University Health Network, Toronto, Canada (32):  P. Manninen, M. Wallace, K. Lukitto, M. Tymianski, P. Porter, F. Gentili, H. El-Beheiry, M. Mosa, P. Mak, M. Balki, S. Shaikh, R. Sawyer, K. Quader, R. Chelliah, P. Berklayd, N. Merah, G. Ghazali, M. McAndrews, J. Ridgley, O. Odukoya, S. Yantha; Wake Forest University Baptist Medical Center, Winston-Salem, North Carolina (31):  J. Wilson, P. Petrozza, C. Miller, K. O'Brien, C. Tong, M. Olympio, J. Reynolds, D. Colonna, S. Glazier, S. Nobles, D. Hill, H. Hulbert, W. Jenkins; Mayo Clinic College of Medicine, Rochester, Minnesota (28):  W. Lanier, D. Piepgras, R. Wilson, F. Meyer, J. Atkinson, M. Link, M. Weglinski, K. Berge, D. McGregor, M. Trenerry, G. Smith, J. Walkes, M. Felmlee-Devine; West Fälische Wilhelms-Universitat Muenster, Muenster, Germany (27):  H. Van Aken, C. Greiner, H. Freise, H. Brors, K. Hahnenkamp, N. Monteiro de Oliveira, C. Schul, D. Moskopp, J. Woelfer, C. Hoenemann, H. Gramke, H. Bone, I. Gibmeier, S. Wirtz, H. Lohmann, J. Freyhoff, B. Bauer; University of Wisconsin Clinical Science Center, Madison, Wisconsin (26):  K. Hogan, R. Dempsey, D. Rusy, B. Badie, B. Iskandar, D. Resnick, P. Deshmukh, J. Fitzpatrick, F. Sasse, T. Broderick, K. Willmann, L. Connery, J. Kish, C. Weasler, N. Page, B. Hermann, J. Jones, D. Dulli, H. Stanko, M. Geraghty, R. Elbe; Montreal Neurologic Hospital, Montreal, Canada (24):  F. Salevsky, R. Leblanc, N. Lapointe, H. MacGregor, D. Sinclair, D. Sirhan, M. Maleki, M. Abou-Madi, D. Chartrand, M. Angle, D. Milovan, Y. Painchaud; Johns Hopkins Medical Institutions, Baltimore, Maryland (23):  M. Mirski, R. Tamargo, S. Rice, A. Olivi, D. Kim, D. Rigamonti, N. Naff, M. Hemstreet, L. Berkow, P. Chery, J. Ulatowski, L. Moore, T. Cunningham, N. McBee, T. Hartman, J. Heidler, A. Hillis, E. Tuffiash, C. Chase, A. Kane, D. Greene-Chandos, M. Torbey, W. Ziai, K. Lane, A. Bhardwaj, N. Subhas; Cleveland Clinic Foundation, Cleveland, Ohio (20):  A. Schubert, M. Mayberg, M. Beven, P. Rasmussen, H. Woo, S. Bhatia, Z. Ebrahim, M. Lotto, F. Vasarhelyi, J. Munis, K. Graves, J. Woletz, G. Chelune, S. Samples, J. Evans, D. Blair, A. Abou-Chebl, F. Shutway, D. Manke, C. Beven; New York Presbyterian Hospital–Weill Medical College of Cornell University, New York, New York (15):  P. Fogarty-Mack, P. Stieg, R. Eliazo, P. Li, H. Riina, C. Lien, L. Ravdin, J. Wang, Y. Kuo; Stanford University Medical Center, Palo Alto, California (15):  R. Jaffe, G. Steinberg, D. Luu, S. Chang, R. Giffard, H. Lemmens, R. Morgan, A. Mathur, M. Angst, A. Meyer, H. Yi, P. Karzmark, T. Bell-Stephens, M. Marcellus; Plymouth Hospitals National Health Service Trust, Plymouth, United Kingdom (14):  J. Sneyd, L. Pobereskin, S. Salsbury, P. Whitfield, R. Sawyer, A. Dashfield, R. Struthers, P. Davies, A. Rushton, V. Petty, S. Harding, E. Richardson; University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (11):  H. Yonas, F. Gyulai, L. Kirby, A. Kassam, N. Bircher, L. Meng, J. Krugh, G. Seever, R. Hendrickson, J. Gebel; Austin Health, Melbourne, Australia (10):  D. Cowie, G. Fabinyi, S. Poustie, G. Davis, A. Drnda, D. Chandrasekara, J. Sturm, T. Phan, A. Shelton, M. Clausen, S. Micallef; Methodist University Hospital, Memphis, Tennessee (8):  A. Sills, F. Steinman, P. Sutton, J. Sanders, D. Van Alstine, D. Leggett, E. Cunningham, W. Hamm, B. Frankel, J. Sorenson, L. Atkins, A. Redmond, S. Dalrymple; University of Alabama at Birmingham, Birmingham, Alabama (7):  S. Black, W. Fisher, C. Hall, D. Wilhite, T. Moore II, P. Blanton, Z. Sha; University of Texas Houston Health Science Center, Houston, Texas (7):  P. Szmuk, D. Kim, A. Ashtari, C. Hagberg, M. Matuszczak, A. Shahen, O. Moise, D. Novy, R. Govindaraj; University of Colorado Health Science Center, Denver, Colorado (4):  L. Jameson, R. Breeze, I. Awad, R. Mattison, T. Anderson, L. Salvia, M. Mosier; University of Oklahoma Health Science Center, Oklahoma City, Oklahoma (3):  C. Loftus, J. Smith, W. Lilley, B. White, M. Lenaerts.