This study was conducted in People’s Hospital of Deyang City, the First Affiliated Hospital of Wenzhou Medical University and the Third Affiliated Hospital of Wenzhou Medical University. The study protocol was approved by the Ethics Committee at the participating hospitals. Written informed consent was obtained from each patient prior to study enrollment or from their legal representative if the patient had lost the capacity to give informed consent.
Between October 2011 and September 2014, we registered 1542 patients who had suffered their first-ever stroke and were admitted to above two participating hospitals. Data were recorded at the time of assessment using a standardized structured form. Detailed methods for data collection of the patients were described in our previous articles [14, 21]. In this study, we enrolled patients who were admitted within 48 hour of the onset of stroke, and suffered from massive MCAI, which was defined as > 50% of the MCA territory on early cranial computed tomography (CT) or magnetic resonance imaging (MRI) scans, with or without the involvement of the adjacent territories [14, 22]. The patients with incomplete hospital records or missing imaging, preexisting score of more than 2 on the modified Rankin scale (mRS), patients included in clinical trials or treated with thrombectomy, and unwilling to participate in this study were excluded. According to aforementioned inclusion criteria and exclusion criteria, a total of 256 patients with massive MCA infarction were enrolled, the detailed data cleaning procedure was presented in Figure 1.
All enrolled patients had an initial brain CT scan at admission. Early signs of cerebral infarction on CT included: (1) the presence of focal hypodensity consistent with the clinical findings; (2) obscuration of the lenticular nucleus or the cortex, or mass effect with effacement of the cortical sulci (grade I); (3) ventricular asymmetry (grade II); (3) shifting of the structures of the median line (grade III) . For the extent of MCAI, Alberta Stroke Program Early CT Score (ASPECTS) was evaluated in the first CT examination . A second CT or MRI was performed between 3 days and 7 days of hospitalization to measure the infarct volume. The infarct volume was determined using the formula 0.5×a×b×c (where a and b are the largest perpendicular diameters measured and c is the sum of slices multiplied with thickness on CT or diffusion-weighted imaging on MRI). An additional CT scan was performed to determine brain edema or hemorrhagic transformation whenever patients had neurological deterioration. Assessment of brain CT or MRI was conducted by a neuroradiologist who was blinded to clinical picture.
For each patient, stroke severity was assessed using National Institutes of Health Stroke Scale (NIHSS) by a member of stroke team on admission, and subsequently on a daily during the period of hospitalization. Age, sex, onset to admission time, baseline systolic and diastolic blood pressure were recorded. Vascular risk factors were investigated. Serum glucose, triglycerides (TG), total plasma cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were measured. Dyslipidemia was defined as TG > 180 mg/dL, TC > 200 mg/dL or use of lipid-lowering medication . Stroke subtypes were classified as atherothrombosis, cardioembolism, stroke of other determined etiology, and stroke of undetermined etiology according to the new subtype classification criteria . All enrolled patients received standard therapies based on standard guidelines .
Measurement of plasma 20-HETE, EETs and DiHETEs levels
Whole blood (4ml) was drawn from each patient at adimission. Plasma was isolated following centrifugation and samples were stored at −80 ◦C until analysis. Total plasma EETs and DiHETEs levels were measured using a stable isotope dilution GC/MS following base hydrolysis and separation on high performance liquid chromatography (HPLC), and plasma 20-HETE level was analyzed using a stable isotope dilution gas chromatography/mass spectrometer (GC/MS), as described in our previous studies [19, 20].
Assessment of m-MCAI and clinical outcome
The primary outcome of this study was m-MCAI. m-MCAI was diagnosed according to the following criteria [13, 26]: massive MCAI showed on follow-up CT including more than two-thirds space-occupying MCAI with midline shift and compression of the basal cisterns, and further consciousness status declined at least 1 point of consciousness item described in the NIHSS compared with the baseline consciousness status on admission, or deterioration of neurological status with clinical signs of uncal herniation and mass effect leading to early death or DHC.
All patients were followed up at 3 months after stroke onset by a certified stroke team member using questionnaires via telephone interview. The secondary outcomes were vascular death and modified Rankin Scale (mRS) at 3 months. Vascular death was defined as vascular mortality due to coronary heart disease, ischemic stroke, or other vascular causes. A favorable outcome was considered as mRS ≤ 2 points, while mRS >2 points was defined as a unfavorable outcome, as described in our previous studies [14, 19- 21].
Previous studies have shown prevalence of m-MCAI to be approximately 30% - 50% in patients with massive MCAI [2-4]. According to this estimate, we expected a minimum sample size requirement of 250 patients with massive MCAI for determining the difference in prevalence of m-MCAI within ± 15% with 95% confidence intervals (CI) [20, 27].
The results are expressed as percentages for categorical variables, and continuous variables are expressed as mean ± Standard Deviation. Baseline and clinical characteristics were compared using χ2 test or Fisher exact test (categorical variables) and the Student t test (continuous variables) between patients with and without m-MCAI. We calculated 20-HETE by quartiles of increasing levels to evaluate for possible threshold effects for m-MCAI.
Multiple logistic regression analysis was used to assess the possible contributing factors for m-MCAI using variables with P values < 0.05 in univariate analysis, and reported as odds ratio (OR) with the 95% confidence intervals (CI). Furthermore, Cox proportional hazard model was performed to account for the probability of m-MCAI according to the 20-HETE levels levels, and reported using Kaplan-Maier curve.
All statistical analyses were performed using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). All tests were two sided, and the threshold level of P < 0.05 was defined as statistical significance.