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Serum neuron specific enolase level as a predictor of prognosis in acute ischemic stroke patients after intravenous thrombolysis
Journal of the Neurological Sciences 359 (2015) 202–206
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Serum neuron specific enolase level as a predictor of prognosis in acute ischemic stroke patients after intravenous thrombolysis Kaili Lu, Xiaofeng Xu, Shasha Cui, Feng Wang, Bin Zhang, Yuwu Zhao ⁎ Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No. 600, Yi Shan Road, Shanghai 200233, PR China
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 4 October 2015 Accepted 17 October 2015 Available online 21 October 2015 Keywords: Acute ischemic stroke Neuron specific enolase Intravenous thrombolysis Stroke severity Favorable outcome Intracranial hemorrhage
a b s t r a c t Objective: Serum neuron specific enolase (NSE) concentrations are significantly correlated with stroke severity and clinical outcome in ischemic stroke patients. We aimed to determine whether the serum levels of neuron specific enolase in acute ischemic stroke (AIS) patients after intravenous thrombolysis are associated with stroke severity, and indicative of favorable outcome. Methods: We prospectively analyzed the serum neuron specific enolase levels with for 67 subjects with AIS patients treated with intravenous recombinant tissue type plasminogen activator (rtPA) within 4.5 h from symptom onset. Neurologic deficit was assessed by the National Institutes of Health Stroke Scale. Clinical outcome was assessed after 90 days according to the modified Rankin Scale. Results: Neuron specific enolase levels correlated with National Institutes of Health Stroke Scale score 24 h after rtPA bolus (R = 0.342, p = 0.005). Regarding the 67 included patients, 32 (47.8%) reached favorable outcome. They had a lower NIHSS score on admission (p = 0.000) and at 24 h after rtPA bolus (p = 0.000), and had lower levels of neuron specific enolase (p = 0.006). But only NIHSS score at 24 h after rtPA bolus rather than neuron specific enolase level was an independent predictor for favorable outcome. Conclusion: We found that after treatment with intravenous rtPA therapy, lower serum neuron specific enolase levels were associated with favorable outcome, which may be confounded by the link to NIHSS score. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Intravenous thrombolytic therapy with recombinant tissue type plasminogen activator (rtPA) improves clinical outcome after acute ischemic stroke within 4.5 h after symptom onset [1,2]. Previous studies have shown that severe stroke indicated by higher NIHSS score and larger infarct volume is a prognostic factor for unfavorable clinical outcome in patients with acute ischemic stroke treated with intravenous rtPA [3,4]. However, reperfusion of ischemic penumbral tissue has been considered as a good surrogate end point in response to intravenous rtPA in previous studies [5,6]. Despite advances in research during the past decades, predictors of clinical outcome represented by clinical data and advanced imaging characteristics remain inconclusive. Recently, neuro-biochemical markers have brought further insights to identify of stroke patients with severe neurological deficit and to predict clinical outcome after rtPA therapy. Neuron specific enolase (NSE) is the marker of brain damage that has been studied most often and mainly used for traumatic
⁎ Corresponding author. E-mail address: [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.jns.2015.10.034 0022-510X/© 2015 Elsevier B.V. All rights reserved.
brain injury [7], stroke [8], and hypoxic encephalopathy [9]. NSE is the γγ-isoenzyme of the glycolytic enzyme enolase found mainly in the cytoplasm of neurons and cells of neuroendocrine origin [10]. When the plasma membrane is impaired functionally or structurally, NSE is released from damaged neurons [7–11]. Numerous studies focusing on NSE have been performed in acute ischemic stroke. Experimental studies that the NSE level increased in the middle cerebral artery occluded models and correlated positively with the volume of infarcted tissue [10]. Accordingly, clinical studies reported increased NSE serum levels in ischemic stroke patients and concluded that increased NSE concentrations are significantly correlated with volumes of infarcted brain areas, severity in acute ischemic stroke measured by NIHSS score, and poor functional outcome [10,12–14]. Previous studies have demonstrated that as the cells in the ischemic penumbra underwent necrosis, NSE levels changed dynamically after symptom onset [15]. In addition, lower NSE levels are associated with clinical–diffusion mismatch [16], a surrogate of salvageable ischemic tissue, which may be more likely to benefit from rtPA. Thus, we aimed to determine the association of serum levels of neuron specific enolase 24 h after intravenous thrombolysis in acute ischemic stroke patients with functional outcome.
K. Lu et al. / Journal of the Neurological Sciences 359 (2015) 202–206
2. Methods 2.1. Participants This was a prospective study targeting consecutive patients with acute ischemic stroke treated with rtPA within the first 4.5 h after symptom onset between August 2013 and June 2015. Stroke onset was defined as the last time the patient was known to be without any neurological deficit. Inclusion and exclusion criteria for intravenous rtPA were used in accordance with those used in the ECASS III [1]. Eligible patients received 0.9 mg of alteplase (Actilyse, Boehringer Ingelheim, Ingelheim am Rhein, Germany) per kilogram, administered intravenously (with an upper limit of 90 mg). Informed consent was obtained from all patients or their next of kin. The study protocol was approved by the local ethics committee. 2.2. Measures 2.2.1. Demographic and medical history On arrival to the emergency department, patients underwent standard neurological examinations, electrocardiogram, blood chemistry, and non-contrast computed tomography (CT). The following clinical data were collected from all patients: 1) patient age and gender; 2) degrees of neurological deficit evaluated by NIHSS score before and 24 h after rtPA infusion; 3) risk factors of stroke including history of hypertension (HTN), and diabetes mellitus (DM), and atrial fibrillation (AF); 4) laboratory parameters including glucose level, HbA1C before rtPA infusion; 5) modified Rankin Scale at 90 days evaluated by certified investigators after rtPA therapy; 6) intracranial hemorrhage at 24 h and mortality at 90 days. 2.2.2. Clinical assessment The modified Rankin scale (mRS) was used to assess clinical outcome at 90 days. Outcome was dichotomized, favorable and unfavorable outcomes at 90 days after therapy were defined as a modified Rankin Scale of 0–1 and 2–6, respectively. And poor outcome with dependency in daily living was defined as a mRS score of 3–6. All patients underwent a CT scan at 24 h or whenever a neurological worsening occurred to evaluate the presence of intracranial hemorrhage (ICH). CT scans were reviewed by a neuroradiologist with extensive experience in acute stroke who was blinded to clinical details and laboratory data. Symptomatic intracranial hemorrhage was defined as any apparently extravascular blood in the brain or within the cranium that was associated with clinical deterioration, as defined by an increase of 4 points or more in NIHSS score, or that led to death and that was identified as the predominant cause of the neurologic deterioration [1]. 2.2.3. Laboratory test All patients had baseline blood samples drawn in the emergency room, to determine baseline glucose levels and HbA1C. Neuronspecific enolase (NSE) levels were measured at 24 h after rtPA infusion. Blood samples, obtained from all patients, were collected in chemistry test tubes. After centrifugation, serum samples were separated, and kept frozen at −80 °C until assayed. Since NSE is also present in erythrocytes, hemolyzed samples were discarded. Serum levels of NSE were measured with commercially available quantitative enzyme-linked immunosorbent assay kits obtained from R&D Systems. Intra- and interassay coefficients of variation were b 3% and b 7%, respectively. And the minimum detection limits were 0.3 ng/mL for NSE. Determinations were performed in a laboratory blinded to clinical data. 2.3. Statistical analysis The analysis was performed with SPSS 16.0 software (SPSS Inc). Continuous variables were described as mean ± SD or median and interquartile range, and compared with Student t test or Mann–Whitney
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U test, as appropriate. Number of patients and percentages for categorical variables were given, and compared using χ2 or Fisher exact test as appropriate. A receiver-operating characteristic curve was applied to determine the cut-point of neuron specific enolase that distinguished between favorable and unfavorable outcome. The Spearman coefficient was applied to verify correlation between examined variables. The relative risks of each variable (with p b 0.1 in the univariate analysis) for favorable outcome were estimated as odds ratios (ORs) in a logistic regression analysis. A level of p b 0.05 was accepted as statistically significant. 3. Results A total of 74 consecutive patients who fulfilled established criteria for intravenous rtPA treatment (0.9 mg/kg) were included in the study. Of these patients, seven were excluded; three patients due to follow-up loss at 90 days after stroke, three without neuron specific enolase examination due to hemolyzed samples, and one due to the lung cancer history. As a result, 67 patients (73.1% male; mean age, 63.6 ± 10.6 years) were enrolled into the present study. The median time from symptom onset to rtPA bolus was 195 min (30 patients ≤3 h, 37 patients between 3 and 4.5 h). Median NIHSS score before intravenous thrombolysis was 8 (range, 2 to 19), and median NIHSS score after thrombolysis 24 h was 4 (range, 0 to 29). The mean neuron specific enolase level of all patients was 15.60 ng/mL (range, 8.48 to 30.69). Serum concentrations of NSE for patients according to baseline characteristics are shown in Fig. 1. Serum concentrations of NSE were significantly higher in patients with AF than those without AF (18.37 ± 4.83 vs. 14.64 ± 4.14 ng/mL; p = 0.003), but no differences were observed with respect to sex, history of hypertension, diabetes mellitus. At 90 days after stroke onset, 32 (47.8%) patients gained favorable outcome group with mRS 0–1 and 35 (52.2%) patients in unfavorable outcome group with mRS 2–6. Of the 12 intracranial hemorrhage (ICH) patients, 3 patient had symptomatic intracranial hemorrhage (sICH) and died within 7 days after thrombolysis, the other 9 patients with intracranial hemorrhage were asymptomatic. Among the 9 patients with death, 3 patient died of sICH and 2 patients died of malignant infarct associated complications in hospital, 1 due to fracture, and 3 due to other causes unrelated to stroke. Serum concentrations of NSE for patients according to different clinical outcomes subgroups are shown in Fig. 2. Compared to respective control group, patients tend to have higher NSE levels in those who had poor outcome (17.36 ± 4.16 vs. 14.66 ± 4.57 ng/mL; p = 0.021), and those who had ICH (18.37 ± 6.24 vs. 14.98 ± 3.96 ng/mL; p = 0.019). But there existed no difference between patients who died at 90 days and those not. Included patients were divided into favorable outcome group and unfavorable outcome group according to mRS at 90 days. Baseline characteristics of the patients in the two groups are shown in Table 1. There were no significant difference between the two groups in terms of age, sex, history of hypertension and atrial fibrillation. In addition, with respect to various parameters of impaired glucose metabolism, including history of diabetes mellitus, baseline glucose, and HbA1C level, no differences were observed between the two groups. However, compared to favorable outcome group, unfavorable outcome patients were more likely to have higher NIHSS score both before (median 10 vs. 4.5; p = 0.000) and 24 h (median 9 vs. 2; p = 0.000) after rtPA. NSE levels were lower in favorable outcome group than in the unfavorable outcome group (14.00 ± 4.26 vs. 17.03 ± 4.45 ng/mL; p = 0.006). The optimal cut-off value to distinguish favorable outcome from unfavorable outcome using a receiver operating characteristics (ROC) curve was 13.90 ng/mL, with sensitivity of 77.1% and specificity of 59.4%. To further estimate the clinical importance of the NSE level, the whole patients were divided into subgroups according to cut-off value of NSE: low NSE group when NSE levels b13.90 ng/mL, and high NSE group when NSE levels ≥ 13.90 ng/mL. Baseline Characteristics and
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Fig. 1. Neuron specific enolase (NSE) levels according to sex (A), history of atrial fibrillation (B), hypertension (C), and diabetes mellitus (D). Values represent mean; error bars represent standard error.
clinical outcomes classified according to NSE subgroups are shown in Table 2. There were no significant differences of neuron specific enolase levels regarding to sex, history of hypertension, history of atrial fibrillation, and history of diabetes mellitus. Patients in the high NSE group less frequently had favorable outcome (32.5% vs. 70.4%; p = 0.002) and more frequently had poor outcome (47.5% vs. 14.8%; p = 0.006) than those in the low NSE group. The age, time from symptom onset to treatment, baseline glucose, and HbA1C level were not different between the two groups. However, there was significant difference in the median NIHSS score 24 h (2 vs. 7, respectively; p = 0.022) after rtPA therapy between the two groups. Neuron specific enolase levels correlated significantly with National Institutes of Health Stroke Scale score at 24 h after rtPA infusion (R = 0.342, p = 0.005) rather than baseline NIHSS score.
We adjusted NIHSS score on admission and ICH at 24 h as variables that had a p b 0.1 in univariate analysis (Table 3), and the OR (odds ratio) of NIHSS score at 24 h after rtPA infusion was 0.662 (95%CI confidence interval 0.488–0.897, p = 0.008) for favorable outcome. However, the adjusted OR of NSE N 13.90 ng/mL (0.229, 95%CI 0.041–1.268, p = 0.091) was not significantly increased for favorable outcome. 4. Discussion NSE has been an important tool in predicting infarct volume, stroke severity and clinical outcome following acute ischemic stroke [13,14]. However, according to several previous studies, NSE did not correlate with neurological deficit and predict clinical outcome [17]. This may
Fig. 2. NSE levels according to favorable outcome (A), poor outcome (B), intracerebral hemorrhage (C), and 90 days mortality (D). Favorable outcome was defined as 90-day modified Rankin Scale ≤ 1. Poor outcome was defined as 90-day modified Rankin Scale ≥3. Values represent mean; error bars represent standard error.
K. Lu et al. / Journal of the Neurological Sciences 359 (2015) 202–206 Table 1 Baseline characteristics of patients with and without favorable outcome (mRS 0–1) at 90 days. Characteristics Age, years Male, n (%) History of hypertension, n (%) History of atrial fibrillation, n (%) History of diabetes mellitus, n (%) NIHSS score On admission 24 h after rtPA infusion NSE (ng/mL) ICH, n (%) Time from symptom onset to treatment (min) Baseline glucose level (mmol/L) HbA1C (%)
mRS (0–1) (n = 32)
mRS (2–6) (n = 35)
p-Value
64.8 ± 12.4 21 (65.6) 22 (68.8) 6 (18.4) 8 (25.0)
62.5 ± 8.6 28 (80.0) 23 (65.7) 11 (31.4) 6 (28.6)
0.387 0.185 0.792 0.234 0.742
4.5 [3–7] 2 [0–3] 14.00 ± 4.26 3 (9.4) 208 [154–238] 7.45 [6.20–9.33] 6.1 [5.7–6.45]
10 [8–13] 9 [7–13.5] 17.03 ± 4.45 9 (25.7) 195 [160–220] 6.90 [6.1–10.35] 5.9 [5.5–7.45]
0.000 0.000 0.006 0.081 0.608 0.547 0.875
Notes—Values are mean ± SD or median [interquartile range] for continuous variables, and number (percentages) for categorical variables. Abbreviations: NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; ICH, intracranial hemorrhage.
partly be explained by the diversity in sampling times, because the NSE level has been shown to change dynamically [12,18]. In addition, for intracranial hemorrhage (ICH) patients, NSE levels were also inconstant and peaked at 24 h, and high serum NSE level was associated with poor outcome [19]. Accounting for the fact that intravenous rtPA may increase the odds of intracranial hemorrhage, probably resulting in increased NSE levels, we measured serum NSE level at 24 h after rtPA bolus when patients underwent cranial computed tomography (CT) to evaluate the presence of intracranial hemorrhage (ICH). In this small sample of acute stroke patients treated with intravenous rtPA, patients with history of AF were more likely to have higher NSE levels, compared to those without AF. This may be explained by previous reports that AF patients tend to have more severe baseline hypo-perfusion, more severe stroke and larger volume of infarcted brain assessed by cranial CT or MRI [20–22], which were significantly correlated with serum NSE concentrations. However, serum NSE levels did not differ significantly between the two groups with respect to the presence of HTN or DM. In contrast, previous studies reported increased Table 2 Baseline characteristics and clinical outcomes in patients classified according to NSE subgroups. Characteristics Age, years Male, n (%) History of hypertension, n (%) History of atrial fibrillation, n (%) History of diabetes mellitus, n (%) NIHSS score On admission 24 h after rt-PA infusion Favorable outcome, n (%) Poor outcome, n (%) ICH, n (%) death, n (%) Time from symptom onset to treatment (min) Baseline glucose level (mmol/L) HbA1C (%)
NSE b 13.9 (n = 27)
NSE ≥ 13.9 (n = 40)
p-Value
61 ± 12 18 (66.7) 18 (66.7) 4 (14.8) 8 (29.6)
65 ± 9 31 (77.5) 27 (67.5) 13 (32.5) 10 (25.0)
0.139 0.326 0.943 0.103 0.675
7 [4–10] 2 [0–5] 19 (70.4) 4 (14.8) 3 (11.1) 2 (7.4) 195 [135–240] 7.3 [6.1–9.5] 6.1 [5.7–6.5]
8.5 [5–11] 7 [2–11] 13 (32.5) 19 (47.5) 9 (22.5) 7 (17.5) 195 [163–223] 6.9 [6.1–9.6] 5.9 [5.5–6.9]
0.278 0.022 0.002 0.006 0.386 0.410 0.906 0.678 0.391
Notes—Values are mean ± SD or median [interquartile range] for continuous variables, and number (percentages) for categorical variables. Abbreviations: NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; favorable outcome, mRS (0–1); poor outcome, mRS (3–6); ICH, intracranial hemorrhage.
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Table 3 Relative risks of variables for favorable outcome (mRS 0–1) at 90 days in patients with intravenous thrombolysis treatment. Variable
OR
95%CI
p value
NIHSS score on admission NIHSS score at 24 h after rtPA infusion ICH NSE N 13.90 ng/mL
0.834 0.662 1.453 0.229
0.601–1.158 0.488–0.897 0.167–12.635 0.041–1.268
0.279 0.008 0.735 0.091
Notes—Abbreviations: CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; favorable outcome, mRS (0–1); ICH, intracranial hemorrhage; OR, odds ratio.
level of serum enolase and mRNA in diabetic patients with and without peripheral neuropathies [23,24], and hypertensive patients [25]. This discrepancy is most likely attributed to different subject selection that others targeted those with no clinical evidence of neurological disease, while we focused on patients with stroke which greatly affected the level of serum enolase, possibly obscuring the slight influence of HTN or DM. Our study showed that lower NSE levels and less neurologic deficit on admission and 24 h after rtPA predicted favorable outcome at 90 days. The clinical predictors of favorable outcome are in accordance with those reported in previous studies [4]. Although higher glucose levels or HbA1C level [26–28], or history of DM [29,30] are considered as important parameters for diagnosing unfavorable outcome and ICH whether with thrombolysis treatment or not, in this study we did not find a correlation between the parameters of impaired glucose metabolism and unfavorable outcome. The inconsistency may be explained by the fact that the median HbA1C level, a well-established marker for long-term elevated glucose level and diabetic vascular damage, was relatively low in both favorable and unfavorable groups (6.1% vs. 5.9%, respectively), which may be attributed to effective control of hyperglycemia with insulin and oral antidiabetic drug. The results of this study showed that NSE serum concentrations at 24 h after rtPA bolus were highly correlated with the severity of the corresponding neurological deficit as quantified by the NIHSS score at 24 h (R = 0.342, p = 0.005). In line with this, previous studies focusing on stroke patients without rtPA suggested that the initial NSE level positively associated with the degree of neurological deficit [14]. Though more detailed studies are needed to further confirm, one study reported that NSE values did not differ between patients who underwent intravenous thrombolysis or conservative medical treatment [12]. There are several explanations for the above association between serum NSE level and NIHSS score in patients with rtPA therapy. Previous studies have demonstrated that as the cells in the ischemic penumbra underwent necrosis and blood–brain barrier impaired, NSE, released from the cell, passed from CSF to the peripheral blood levels after symptom onset [11]. In those patients who responded to thrombolysis, the occluded artery recanalized and the ischemic penumbra was reperfused, so the NIHSS score decreased, and the NSE levels stopped increasing. In contrast, in those patients who responded poorly to rtPA without recanalization or reperfusion, the ischemic penumbra involved to the infarct core, so both the NIHSS score and the NSE levels increased. Accordingly, in patients with reperfusion injury or hemorrhagic transformation, ischemia-induced damage and BBB disruption may be aggravated, thus causing more severe neurological deficit and higher NSE levels leaking from damaged BBB. So post-treatment neuron specific enolase levels correlated significantly with NIHSS score at 24 h after rtPA infusion rather than baseline NIHSS score. The division of our sample by the cutoff point of NSE level gives the impression that patients in the high NSE group less frequently had favorable outcome, and more frequently gained poor outcome than those in the low NSE group. However, ICH possibility and mortality rate did not show difference between the two groups. This might in large part be explained by the fact that patients treated with rtPA in
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our study have diverse degree of ICH, ranging from slight hemorrhagic transformation to deadly symptomatic intracranial hemorrhage, exhibiting different damage to the brain tissue, causing various increase in NSE level. In addition, among the causes of 9 death at 3 months, 3 patients died of causes unrelated to stroke, which might not be reflected by NSE levels. Correlations between NSE and functional disability after 3 months may contain greater clinical potential. In our study, a NSE threshold of b13.90 ng/mL and a lower NIHSS score 24 h after rtPA infusion can help identify patients likely to reach favorable outcome in univariate analysis. This threshold is quite similar to the result from published data, which showed that acute ischemic stroke patients with NSE concentration above 13 ng/mL tend to have poor functional outcome [8]. Lower NSE levels are associated with clinical–diffusion mismatch [16], which has been suggested as a surrogate of ischemic brain at risk of infarction and might be used to recognize salvageable ischemic tissue [31]. Thus, patients with lower NSE may be more likely to benefit from rtPA. However, included in multivariate analysis, only lower NIHSS score at 24 h after rtPA infusion rather than NSE levels was independently associated with favorable outcome, which might be attributed to the link to NIHSS score at 24 h after rtPA infusion. Some restrictions have to be considered in the present study. First, our data result from hyper-acute ischemic stroke patients with thrombolysis therapy, without the control group. Whether treatment with rtPA, via damaging the blood–brain barrier, increases the serum NSE level has not been confirmed. Thus, the association between the NSE level and stroke severity estimated by NIHSS score may have been over-estimated. Second, our sample size was too small to confirm the validity of our conclusions. Furthermore, NSE is vulnerable to hemolysis and therefore the interpretation of hemolytic samples is not possible. In summary, our results suggest that NSE is associated with patient's clinical deficits assessed by NIHSS score, and may be used as an additional predictor of functional outcome after thrombolysis treatment in AIS patients. More detailed studies in addition to this study are necessary to further ascertain this hypothesis. Acknowledgments The authors wish to thank the staff of our stroke unit for extensive collaboration. References [1] W. Hacke, M. Kaste, E. Bluhmki, M. Brozman, A. Davalos, D. Guidetti, et al., Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke, N. Engl. J. Med. 359 (2008) 1317–1329. [2] J. Emberson, K.R. Lees, P. Lyden, L. Blackwell, G. Albers, E. Bluhmki, et al., Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials, Lancet 384 (2014) 1929–1935. [3] A. Kruetzelmann, M. Kohrmann, J. Sobesky, B. Cheng, M. Rosenkranz, J. Rother, et al., Pretreatment diffusion-weighted imaging lesion volume predicts favorable outcome after intravenous thrombolysis with tissue-type plasminogen activator in acute ischemic stroke, Stroke 42 (2011) 1251–1254. [4] D. Sanak, R. Herzig, J. Zapletalova, D. Horak, M. Kral, D. Skoloudik, et al., Predictors of good clinical outcome in acute stroke patients treated with intravenous thrombolysis, Acta Neurol. Scand. 123 (2011) 339–344. [5] S.M. Davis, G.A. Donnan, M.W. Parsons, C. Levi, K.S. Butcher, A. Peeters, et al., Effects of alteplase beyond 3 h after stroke in the Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET): a placebo-controlled randomised trial, Lancet Neurol. 7 (2008) 299–309. [6] G.W. Albers, V.N. Thijs, L. Wechsler, S. Kemp, G. Schlaug, E. Skalabrin, et al., Magnetic resonance imaging profiles predict clinical response to early reperfusion: the diffusion and perfusion imaging evaluation for understanding stroke evolution (DEFUSE) study, Ann. Neurol. 60 (2006) 508–517.
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Serum neuron specific enolase level as a predictor of prognosis in acute ischemic stroke patients after intravenous thrombolysis Kaili Lu, Xiaofeng Xu, Shasha Cui, Feng Wang, Bin Zhang, Yuwu Zhao ⁎ Department of Neurology, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, No. 600, Yi Shan Road, Shanghai 200233, PR China
a r t i c l e
i n f o
Article history: Received 16 July 2015 Received in revised form 4 October 2015 Accepted 17 October 2015 Available online 21 October 2015 Keywords: Acute ischemic stroke Neuron specific enolase Intravenous thrombolysis Stroke severity Favorable outcome Intracranial hemorrhage
a b s t r a c t Objective: Serum neuron specific enolase (NSE) concentrations are significantly correlated with stroke severity and clinical outcome in ischemic stroke patients. We aimed to determine whether the serum levels of neuron specific enolase in acute ischemic stroke (AIS) patients after intravenous thrombolysis are associated with stroke severity, and indicative of favorable outcome. Methods: We prospectively analyzed the serum neuron specific enolase levels with for 67 subjects with AIS patients treated with intravenous recombinant tissue type plasminogen activator (rtPA) within 4.5 h from symptom onset. Neurologic deficit was assessed by the National Institutes of Health Stroke Scale. Clinical outcome was assessed after 90 days according to the modified Rankin Scale. Results: Neuron specific enolase levels correlated with National Institutes of Health Stroke Scale score 24 h after rtPA bolus (R = 0.342, p = 0.005). Regarding the 67 included patients, 32 (47.8%) reached favorable outcome. They had a lower NIHSS score on admission (p = 0.000) and at 24 h after rtPA bolus (p = 0.000), and had lower levels of neuron specific enolase (p = 0.006). But only NIHSS score at 24 h after rtPA bolus rather than neuron specific enolase level was an independent predictor for favorable outcome. Conclusion: We found that after treatment with intravenous rtPA therapy, lower serum neuron specific enolase levels were associated with favorable outcome, which may be confounded by the link to NIHSS score. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Intravenous thrombolytic therapy with recombinant tissue type plasminogen activator (rtPA) improves clinical outcome after acute ischemic stroke within 4.5 h after symptom onset [1,2]. Previous studies have shown that severe stroke indicated by higher NIHSS score and larger infarct volume is a prognostic factor for unfavorable clinical outcome in patients with acute ischemic stroke treated with intravenous rtPA [3,4]. However, reperfusion of ischemic penumbral tissue has been considered as a good surrogate end point in response to intravenous rtPA in previous studies [5,6]. Despite advances in research during the past decades, predictors of clinical outcome represented by clinical data and advanced imaging characteristics remain inconclusive. Recently, neuro-biochemical markers have brought further insights to identify of stroke patients with severe neurological deficit and to predict clinical outcome after rtPA therapy. Neuron specific enolase (NSE) is the marker of brain damage that has been studied most often and mainly used for traumatic
⁎ Corresponding author. E-mail address: [email protected] (Y. Zhao).
http://dx.doi.org/10.1016/j.jns.2015.10.034 0022-510X/© 2015 Elsevier B.V. All rights reserved.
brain injury [7], stroke [8], and hypoxic encephalopathy [9]. NSE is the γγ-isoenzyme of the glycolytic enzyme enolase found mainly in the cytoplasm of neurons and cells of neuroendocrine origin [10]. When the plasma membrane is impaired functionally or structurally, NSE is released from damaged neurons [7–11]. Numerous studies focusing on NSE have been performed in acute ischemic stroke. Experimental studies that the NSE level increased in the middle cerebral artery occluded models and correlated positively with the volume of infarcted tissue [10]. Accordingly, clinical studies reported increased NSE serum levels in ischemic stroke patients and concluded that increased NSE concentrations are significantly correlated with volumes of infarcted brain areas, severity in acute ischemic stroke measured by NIHSS score, and poor functional outcome [10,12–14]. Previous studies have demonstrated that as the cells in the ischemic penumbra underwent necrosis, NSE levels changed dynamically after symptom onset [15]. In addition, lower NSE levels are associated with clinical–diffusion mismatch [16], a surrogate of salvageable ischemic tissue, which may be more likely to benefit from rtPA. Thus, we aimed to determine the association of serum levels of neuron specific enolase 24 h after intravenous thrombolysis in acute ischemic stroke patients with functional outcome.
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2. Methods 2.1. Participants This was a prospective study targeting consecutive patients with acute ischemic stroke treated with rtPA within the first 4.5 h after symptom onset between August 2013 and June 2015. Stroke onset was defined as the last time the patient was known to be without any neurological deficit. Inclusion and exclusion criteria for intravenous rtPA were used in accordance with those used in the ECASS III [1]. Eligible patients received 0.9 mg of alteplase (Actilyse, Boehringer Ingelheim, Ingelheim am Rhein, Germany) per kilogram, administered intravenously (with an upper limit of 90 mg). Informed consent was obtained from all patients or their next of kin. The study protocol was approved by the local ethics committee. 2.2. Measures 2.2.1. Demographic and medical history On arrival to the emergency department, patients underwent standard neurological examinations, electrocardiogram, blood chemistry, and non-contrast computed tomography (CT). The following clinical data were collected from all patients: 1) patient age and gender; 2) degrees of neurological deficit evaluated by NIHSS score before and 24 h after rtPA infusion; 3) risk factors of stroke including history of hypertension (HTN), and diabetes mellitus (DM), and atrial fibrillation (AF); 4) laboratory parameters including glucose level, HbA1C before rtPA infusion; 5) modified Rankin Scale at 90 days evaluated by certified investigators after rtPA therapy; 6) intracranial hemorrhage at 24 h and mortality at 90 days. 2.2.2. Clinical assessment The modified Rankin scale (mRS) was used to assess clinical outcome at 90 days. Outcome was dichotomized, favorable and unfavorable outcomes at 90 days after therapy were defined as a modified Rankin Scale of 0–1 and 2–6, respectively. And poor outcome with dependency in daily living was defined as a mRS score of 3–6. All patients underwent a CT scan at 24 h or whenever a neurological worsening occurred to evaluate the presence of intracranial hemorrhage (ICH). CT scans were reviewed by a neuroradiologist with extensive experience in acute stroke who was blinded to clinical details and laboratory data. Symptomatic intracranial hemorrhage was defined as any apparently extravascular blood in the brain or within the cranium that was associated with clinical deterioration, as defined by an increase of 4 points or more in NIHSS score, or that led to death and that was identified as the predominant cause of the neurologic deterioration [1]. 2.2.3. Laboratory test All patients had baseline blood samples drawn in the emergency room, to determine baseline glucose levels and HbA1C. Neuronspecific enolase (NSE) levels were measured at 24 h after rtPA infusion. Blood samples, obtained from all patients, were collected in chemistry test tubes. After centrifugation, serum samples were separated, and kept frozen at −80 °C until assayed. Since NSE is also present in erythrocytes, hemolyzed samples were discarded. Serum levels of NSE were measured with commercially available quantitative enzyme-linked immunosorbent assay kits obtained from R&D Systems. Intra- and interassay coefficients of variation were b 3% and b 7%, respectively. And the minimum detection limits were 0.3 ng/mL for NSE. Determinations were performed in a laboratory blinded to clinical data. 2.3. Statistical analysis The analysis was performed with SPSS 16.0 software (SPSS Inc). Continuous variables were described as mean ± SD or median and interquartile range, and compared with Student t test or Mann–Whitney
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U test, as appropriate. Number of patients and percentages for categorical variables were given, and compared using χ2 or Fisher exact test as appropriate. A receiver-operating characteristic curve was applied to determine the cut-point of neuron specific enolase that distinguished between favorable and unfavorable outcome. The Spearman coefficient was applied to verify correlation between examined variables. The relative risks of each variable (with p b 0.1 in the univariate analysis) for favorable outcome were estimated as odds ratios (ORs) in a logistic regression analysis. A level of p b 0.05 was accepted as statistically significant. 3. Results A total of 74 consecutive patients who fulfilled established criteria for intravenous rtPA treatment (0.9 mg/kg) were included in the study. Of these patients, seven were excluded; three patients due to follow-up loss at 90 days after stroke, three without neuron specific enolase examination due to hemolyzed samples, and one due to the lung cancer history. As a result, 67 patients (73.1% male; mean age, 63.6 ± 10.6 years) were enrolled into the present study. The median time from symptom onset to rtPA bolus was 195 min (30 patients ≤3 h, 37 patients between 3 and 4.5 h). Median NIHSS score before intravenous thrombolysis was 8 (range, 2 to 19), and median NIHSS score after thrombolysis 24 h was 4 (range, 0 to 29). The mean neuron specific enolase level of all patients was 15.60 ng/mL (range, 8.48 to 30.69). Serum concentrations of NSE for patients according to baseline characteristics are shown in Fig. 1. Serum concentrations of NSE were significantly higher in patients with AF than those without AF (18.37 ± 4.83 vs. 14.64 ± 4.14 ng/mL; p = 0.003), but no differences were observed with respect to sex, history of hypertension, diabetes mellitus. At 90 days after stroke onset, 32 (47.8%) patients gained favorable outcome group with mRS 0–1 and 35 (52.2%) patients in unfavorable outcome group with mRS 2–6. Of the 12 intracranial hemorrhage (ICH) patients, 3 patient had symptomatic intracranial hemorrhage (sICH) and died within 7 days after thrombolysis, the other 9 patients with intracranial hemorrhage were asymptomatic. Among the 9 patients with death, 3 patient died of sICH and 2 patients died of malignant infarct associated complications in hospital, 1 due to fracture, and 3 due to other causes unrelated to stroke. Serum concentrations of NSE for patients according to different clinical outcomes subgroups are shown in Fig. 2. Compared to respective control group, patients tend to have higher NSE levels in those who had poor outcome (17.36 ± 4.16 vs. 14.66 ± 4.57 ng/mL; p = 0.021), and those who had ICH (18.37 ± 6.24 vs. 14.98 ± 3.96 ng/mL; p = 0.019). But there existed no difference between patients who died at 90 days and those not. Included patients were divided into favorable outcome group and unfavorable outcome group according to mRS at 90 days. Baseline characteristics of the patients in the two groups are shown in Table 1. There were no significant difference between the two groups in terms of age, sex, history of hypertension and atrial fibrillation. In addition, with respect to various parameters of impaired glucose metabolism, including history of diabetes mellitus, baseline glucose, and HbA1C level, no differences were observed between the two groups. However, compared to favorable outcome group, unfavorable outcome patients were more likely to have higher NIHSS score both before (median 10 vs. 4.5; p = 0.000) and 24 h (median 9 vs. 2; p = 0.000) after rtPA. NSE levels were lower in favorable outcome group than in the unfavorable outcome group (14.00 ± 4.26 vs. 17.03 ± 4.45 ng/mL; p = 0.006). The optimal cut-off value to distinguish favorable outcome from unfavorable outcome using a receiver operating characteristics (ROC) curve was 13.90 ng/mL, with sensitivity of 77.1% and specificity of 59.4%. To further estimate the clinical importance of the NSE level, the whole patients were divided into subgroups according to cut-off value of NSE: low NSE group when NSE levels b13.90 ng/mL, and high NSE group when NSE levels ≥ 13.90 ng/mL. Baseline Characteristics and
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Fig. 1. Neuron specific enolase (NSE) levels according to sex (A), history of atrial fibrillation (B), hypertension (C), and diabetes mellitus (D). Values represent mean; error bars represent standard error.
clinical outcomes classified according to NSE subgroups are shown in Table 2. There were no significant differences of neuron specific enolase levels regarding to sex, history of hypertension, history of atrial fibrillation, and history of diabetes mellitus. Patients in the high NSE group less frequently had favorable outcome (32.5% vs. 70.4%; p = 0.002) and more frequently had poor outcome (47.5% vs. 14.8%; p = 0.006) than those in the low NSE group. The age, time from symptom onset to treatment, baseline glucose, and HbA1C level were not different between the two groups. However, there was significant difference in the median NIHSS score 24 h (2 vs. 7, respectively; p = 0.022) after rtPA therapy between the two groups. Neuron specific enolase levels correlated significantly with National Institutes of Health Stroke Scale score at 24 h after rtPA infusion (R = 0.342, p = 0.005) rather than baseline NIHSS score.
We adjusted NIHSS score on admission and ICH at 24 h as variables that had a p b 0.1 in univariate analysis (Table 3), and the OR (odds ratio) of NIHSS score at 24 h after rtPA infusion was 0.662 (95%CI confidence interval 0.488–0.897, p = 0.008) for favorable outcome. However, the adjusted OR of NSE N 13.90 ng/mL (0.229, 95%CI 0.041–1.268, p = 0.091) was not significantly increased for favorable outcome. 4. Discussion NSE has been an important tool in predicting infarct volume, stroke severity and clinical outcome following acute ischemic stroke [13,14]. However, according to several previous studies, NSE did not correlate with neurological deficit and predict clinical outcome [17]. This may
Fig. 2. NSE levels according to favorable outcome (A), poor outcome (B), intracerebral hemorrhage (C), and 90 days mortality (D). Favorable outcome was defined as 90-day modified Rankin Scale ≤ 1. Poor outcome was defined as 90-day modified Rankin Scale ≥3. Values represent mean; error bars represent standard error.
K. Lu et al. / Journal of the Neurological Sciences 359 (2015) 202–206 Table 1 Baseline characteristics of patients with and without favorable outcome (mRS 0–1) at 90 days. Characteristics Age, years Male, n (%) History of hypertension, n (%) History of atrial fibrillation, n (%) History of diabetes mellitus, n (%) NIHSS score On admission 24 h after rtPA infusion NSE (ng/mL) ICH, n (%) Time from symptom onset to treatment (min) Baseline glucose level (mmol/L) HbA1C (%)
mRS (0–1) (n = 32)
mRS (2–6) (n = 35)
p-Value
64.8 ± 12.4 21 (65.6) 22 (68.8) 6 (18.4) 8 (25.0)
62.5 ± 8.6 28 (80.0) 23 (65.7) 11 (31.4) 6 (28.6)
0.387 0.185 0.792 0.234 0.742
4.5 [3–7] 2 [0–3] 14.00 ± 4.26 3 (9.4) 208 [154–238] 7.45 [6.20–9.33] 6.1 [5.7–6.45]
10 [8–13] 9 [7–13.5] 17.03 ± 4.45 9 (25.7) 195 [160–220] 6.90 [6.1–10.35] 5.9 [5.5–7.45]
0.000 0.000 0.006 0.081 0.608 0.547 0.875
Notes—Values are mean ± SD or median [interquartile range] for continuous variables, and number (percentages) for categorical variables. Abbreviations: NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; ICH, intracranial hemorrhage.
partly be explained by the diversity in sampling times, because the NSE level has been shown to change dynamically [12,18]. In addition, for intracranial hemorrhage (ICH) patients, NSE levels were also inconstant and peaked at 24 h, and high serum NSE level was associated with poor outcome [19]. Accounting for the fact that intravenous rtPA may increase the odds of intracranial hemorrhage, probably resulting in increased NSE levels, we measured serum NSE level at 24 h after rtPA bolus when patients underwent cranial computed tomography (CT) to evaluate the presence of intracranial hemorrhage (ICH). In this small sample of acute stroke patients treated with intravenous rtPA, patients with history of AF were more likely to have higher NSE levels, compared to those without AF. This may be explained by previous reports that AF patients tend to have more severe baseline hypo-perfusion, more severe stroke and larger volume of infarcted brain assessed by cranial CT or MRI [20–22], which were significantly correlated with serum NSE concentrations. However, serum NSE levels did not differ significantly between the two groups with respect to the presence of HTN or DM. In contrast, previous studies reported increased Table 2 Baseline characteristics and clinical outcomes in patients classified according to NSE subgroups. Characteristics Age, years Male, n (%) History of hypertension, n (%) History of atrial fibrillation, n (%) History of diabetes mellitus, n (%) NIHSS score On admission 24 h after rt-PA infusion Favorable outcome, n (%) Poor outcome, n (%) ICH, n (%) death, n (%) Time from symptom onset to treatment (min) Baseline glucose level (mmol/L) HbA1C (%)
NSE b 13.9 (n = 27)
NSE ≥ 13.9 (n = 40)
p-Value
61 ± 12 18 (66.7) 18 (66.7) 4 (14.8) 8 (29.6)
65 ± 9 31 (77.5) 27 (67.5) 13 (32.5) 10 (25.0)
0.139 0.326 0.943 0.103 0.675
7 [4–10] 2 [0–5] 19 (70.4) 4 (14.8) 3 (11.1) 2 (7.4) 195 [135–240] 7.3 [6.1–9.5] 6.1 [5.7–6.5]
8.5 [5–11] 7 [2–11] 13 (32.5) 19 (47.5) 9 (22.5) 7 (17.5) 195 [163–223] 6.9 [6.1–9.6] 5.9 [5.5–6.9]
0.278 0.022 0.002 0.006 0.386 0.410 0.906 0.678 0.391
Notes—Values are mean ± SD or median [interquartile range] for continuous variables, and number (percentages) for categorical variables. Abbreviations: NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; favorable outcome, mRS (0–1); poor outcome, mRS (3–6); ICH, intracranial hemorrhage.
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Table 3 Relative risks of variables for favorable outcome (mRS 0–1) at 90 days in patients with intravenous thrombolysis treatment. Variable
OR
95%CI
p value
NIHSS score on admission NIHSS score at 24 h after rtPA infusion ICH NSE N 13.90 ng/mL
0.834 0.662 1.453 0.229
0.601–1.158 0.488–0.897 0.167–12.635 0.041–1.268
0.279 0.008 0.735 0.091
Notes—Abbreviations: CI, confidence interval; NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale; rtPA, recombinant tissue type plasminogen activator; NSE, neuron specific enolase; favorable outcome, mRS (0–1); ICH, intracranial hemorrhage; OR, odds ratio.
level of serum enolase and mRNA in diabetic patients with and without peripheral neuropathies [23,24], and hypertensive patients [25]. This discrepancy is most likely attributed to different subject selection that others targeted those with no clinical evidence of neurological disease, while we focused on patients with stroke which greatly affected the level of serum enolase, possibly obscuring the slight influence of HTN or DM. Our study showed that lower NSE levels and less neurologic deficit on admission and 24 h after rtPA predicted favorable outcome at 90 days. The clinical predictors of favorable outcome are in accordance with those reported in previous studies [4]. Although higher glucose levels or HbA1C level [26–28], or history of DM [29,30] are considered as important parameters for diagnosing unfavorable outcome and ICH whether with thrombolysis treatment or not, in this study we did not find a correlation between the parameters of impaired glucose metabolism and unfavorable outcome. The inconsistency may be explained by the fact that the median HbA1C level, a well-established marker for long-term elevated glucose level and diabetic vascular damage, was relatively low in both favorable and unfavorable groups (6.1% vs. 5.9%, respectively), which may be attributed to effective control of hyperglycemia with insulin and oral antidiabetic drug. The results of this study showed that NSE serum concentrations at 24 h after rtPA bolus were highly correlated with the severity of the corresponding neurological deficit as quantified by the NIHSS score at 24 h (R = 0.342, p = 0.005). In line with this, previous studies focusing on stroke patients without rtPA suggested that the initial NSE level positively associated with the degree of neurological deficit [14]. Though more detailed studies are needed to further confirm, one study reported that NSE values did not differ between patients who underwent intravenous thrombolysis or conservative medical treatment [12]. There are several explanations for the above association between serum NSE level and NIHSS score in patients with rtPA therapy. Previous studies have demonstrated that as the cells in the ischemic penumbra underwent necrosis and blood–brain barrier impaired, NSE, released from the cell, passed from CSF to the peripheral blood levels after symptom onset [11]. In those patients who responded to thrombolysis, the occluded artery recanalized and the ischemic penumbra was reperfused, so the NIHSS score decreased, and the NSE levels stopped increasing. In contrast, in those patients who responded poorly to rtPA without recanalization or reperfusion, the ischemic penumbra involved to the infarct core, so both the NIHSS score and the NSE levels increased. Accordingly, in patients with reperfusion injury or hemorrhagic transformation, ischemia-induced damage and BBB disruption may be aggravated, thus causing more severe neurological deficit and higher NSE levels leaking from damaged BBB. So post-treatment neuron specific enolase levels correlated significantly with NIHSS score at 24 h after rtPA infusion rather than baseline NIHSS score. The division of our sample by the cutoff point of NSE level gives the impression that patients in the high NSE group less frequently had favorable outcome, and more frequently gained poor outcome than those in the low NSE group. However, ICH possibility and mortality rate did not show difference between the two groups. This might in large part be explained by the fact that patients treated with rtPA in
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our study have diverse degree of ICH, ranging from slight hemorrhagic transformation to deadly symptomatic intracranial hemorrhage, exhibiting different damage to the brain tissue, causing various increase in NSE level. In addition, among the causes of 9 death at 3 months, 3 patients died of causes unrelated to stroke, which might not be reflected by NSE levels. Correlations between NSE and functional disability after 3 months may contain greater clinical potential. In our study, a NSE threshold of b13.90 ng/mL and a lower NIHSS score 24 h after rtPA infusion can help identify patients likely to reach favorable outcome in univariate analysis. This threshold is quite similar to the result from published data, which showed that acute ischemic stroke patients with NSE concentration above 13 ng/mL tend to have poor functional outcome [8]. Lower NSE levels are associated with clinical–diffusion mismatch [16], which has been suggested as a surrogate of ischemic brain at risk of infarction and might be used to recognize salvageable ischemic tissue [31]. Thus, patients with lower NSE may be more likely to benefit from rtPA. However, included in multivariate analysis, only lower NIHSS score at 24 h after rtPA infusion rather than NSE levels was independently associated with favorable outcome, which might be attributed to the link to NIHSS score at 24 h after rtPA infusion. Some restrictions have to be considered in the present study. First, our data result from hyper-acute ischemic stroke patients with thrombolysis therapy, without the control group. Whether treatment with rtPA, via damaging the blood–brain barrier, increases the serum NSE level has not been confirmed. Thus, the association between the NSE level and stroke severity estimated by NIHSS score may have been over-estimated. Second, our sample size was too small to confirm the validity of our conclusions. Furthermore, NSE is vulnerable to hemolysis and therefore the interpretation of hemolytic samples is not possible. In summary, our results suggest that NSE is associated with patient's clinical deficits assessed by NIHSS score, and may be used as an additional predictor of functional outcome after thrombolysis treatment in AIS patients. More detailed studies in addition to this study are necessary to further ascertain this hypothesis. Acknowledgments The authors wish to thank the staff of our stroke unit for extensive collaboration. References [1] W. Hacke, M. Kaste, E. Bluhmki, M. Brozman, A. Davalos, D. Guidetti, et al., Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke, N. Engl. J. Med. 359 (2008) 1317–1329. [2] J. Emberson, K.R. Lees, P. Lyden, L. Blackwell, G. Albers, E. Bluhmki, et al., Effect of treatment delay, age, and stroke severity on the effects of intravenous thrombolysis with alteplase for acute ischaemic stroke: a meta-analysis of individual patient data from randomised trials, Lancet 384 (2014) 1929–1935. [3] A. Kruetzelmann, M. Kohrmann, J. Sobesky, B. Cheng, M. Rosenkranz, J. Rother, et al., Pretreatment diffusion-weighted imaging lesion volume predicts favorable outcome after intravenous thrombolysis with tissue-type plasminogen activator in acute ischemic stroke, Stroke 42 (2011) 1251–1254. [4] D. Sanak, R. Herzig, J. Zapletalova, D. Horak, M. Kral, D. Skoloudik, et al., Predictors of good clinical outcome in acute stroke patients treated with intravenous thrombolysis, Acta Neurol. Scand. 123 (2011) 339–344. [5] S.M. Davis, G.A. Donnan, M.W. Parsons, C. Levi, K.S. Butcher, A. Peeters, et al., Effects of alteplase beyond 3 h after stroke in the Echoplanar Imaging Thrombolytic Evaluation Trial (EPITHET): a placebo-controlled randomised trial, Lancet Neurol. 7 (2008) 299–309. [6] G.W. Albers, V.N. Thijs, L. Wechsler, S. Kemp, G. Schlaug, E. Skalabrin, et al., Magnetic resonance imaging profiles predict clinical response to early reperfusion: the diffusion and perfusion imaging evaluation for understanding stroke evolution (DEFUSE) study, Ann. Neurol. 60 (2006) 508–517.
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