Patients were enrolled from January 2017 to October 2019. The selection of participating hospitals was initiated within the neuro-intensive care unit (ICU) section of the Korean Society of Intensive Care Medicine, with the identification of investigators which have thereafter selected any potential ICUs with neurosurgical facilities and an adequate number of patients with aSAH admitted per year.

The inclusion criteria to be enrolled in the present study were as follows: 1) diagnosis of aneurysm rupture SAH based on computed tomography (CT) and CT angiography (CTA); 2) age ≥ 18 or ≤ 80 years; and 3) expectation to stay in the ICU for > 48 hours. The exclusion criteria were as follows: 1) trauma related hemorrhage; 2) patients on medication of antiplatelets (aspirin, clopidogrel, or cilostazol) or anticoagulation (heparin or low-molecular weight heparin, vitamin K antagonist (warfarin) or direct oral anticoagulants); 3) pregnancy; 4) seizure or status epilepticus disorder requiring anticonvulsant medication; and 5) medically ill conditions that may affect the clinical outcome (i.e., liver cirrhosis, post-cardiac arrest, imminent death or do not resuscitate ordered patients and so on).

The primary outcome was a neurological outcome assessed at 30-days using a modified Rankin scale (mRS).27 Secondary outcomes were divided into clinical and radiological values. The clinical outcomes were the time to reach target sedation level (using the Richmond agitation sedation scale [RASS], target of −2 to 0), need to perform tracheostomy, and total duration of hospital and ICU stay. The radiologic outcomes were serial changes in mid-line shifting due to PHE on CT and the incidence of vasospasms.

Eligible patients were randomly assigned to receive either antioxidants or matching placebo solutions. Randomization was performed in a 1:1 ratio, without stratification and using a block size of 4, by means of a computerized central internet-based allocation service based on computer-generated random numbers. Study investigators were aware of the randomization results whereas enrolled patients were blinded to the result of study drug randomization. The antioxidants used were acetylcysteine (2,000 mg [1,000 mg twice daily {BID}] intravenously [IV] per day) and selenium (1,600 µg [800 µg BID] IV per day). The control group received IV placebo. Antioxidants were given as soon as possible on the day of admission (within 24 hours of admission) for 14 days. These intravenous antioxidants were administered in addition to standard nutrition. Serum selenium level (normal range: 5.8–23.4 µg/dL) was checked twice weekly for 14 days. If the selenium level was higher than the upper limit, administration was discontinued and the level was assessed on the next day to determine the administration. There is no available test to check serum acetylcysteine levels.

Relevant data were obtained from the medical records by a neurosurgeon and trained research nurse. The following factors were analyzed: 1) age; 2) sex; 3) comorbidities: hypertension (previous hypertension diagnosis or taking related medication), diabetes mellitus (previous diabetes mellitus diagnosis or taking related medication), hyperlipidemia (total cholesterol ≥ 240 mg/dL, triglyceride ≥ 200 mg/dL, high-density lipoprotein cholesterol ≤ 40 mg/dL, low-density lipoprotein cholesterol ≥ 150 mg/dL, or lipid-lowering medication use), ischemic heart disease (previous diagnosis of angina or myocardial infarction or related medication in use), chronic kidney injury; 4) Hunt and Hess grade28 on admission; 5) treatment modality: microsurgery or endovascular; 6) admission scores (the worst of the first 24 hours) including Glasgow coma scale (GCS), mRS and RASS; 7) time interval between last normal time to hospital admission (hours); 8) baseline blood test results (blood pressure, glucose, total cholesterol, platelets, activated partial thromboplastin time, prothrombin time, and international normalized ratio); 9) use of osmotic agents (mannitol or hypertonic saline); 10) angioplasty for vasospasm; and 11) hydrocephalus on admission. Agitation and sedation levels were examined using the RASS. Our target RASS score was between −2 and 0.

All patients underwent CT and CTA upon admission followed by a transfemoral catheter angiography for treatment decisions (microsurgery vs. endovascular). The collection of radiologic data included: 1) location of ruptured aneurysm (internal carotid artery, anterior cerebral artery, middle cerebral artery, posterior cerebral artery, anterior inferior cerebellar artery, posterior inferior cerebellar artery, and basilar artery); and 2) modified Fisher grade29 for distribution and pattern of SAH.

Serious adverse events (SAEs) were prespecified and had to be reported within 48 hours of identification; 1) pneumonia or ARDS; 2) gastrointestinal bleeding; 3) acute myocardial ischemia or heart failure; 4) cardiac arrest 5) acute renal injury; 6) venous thrombotic events (deep vein thrombosis or pulmonary thromboembolism); 7) sepsis (evidence of an infection and organ failure attributed to it) or septic shock (mean arterial pressure < 65 mmHg despite fluid resuscitation requiring inotropics or vasopressors); and 8) disseminated intravascular coagulation.

To evaluate the radiological impact of antioxidants in aSAH patients, we selected patients according to their hemorrhage distributions (i.e., modified Fisher grade). Since modified Fisher grade 4 patients have localized intracerebral hemorrhage (ICH) or intraventricular hemorrhage with diffuse SAH, we measured the volume of hematoma, PHE and PHE ratio on post-operative day 0, 7 and 14 for these patients. PHE ratio was defined as the differential value between hemorrhage and PHE volume. The diagnosis of vasospasm was dependent on both clinical and radiological basis.3031 CTA, magnetic resonance angiography (MRA) or transcranial doppler (TCD) ultrasonography were adopted for the radiological diagnosis of vasospasm. The definition of vasospasm in the present study were as follow32; 1) symptomatic vasospasm, defined as clinical deterioration deemed secondary to vasospasm after other causes were eliminated; 2) delayed cerebral ischemia, defined as symptomatic vasospasm, or infarction on CT attributable to vasospasm; 3) angiographic spasm, as seen on digital subtraction angiography; and 4) TCD spasm, defined as any mean flow velocity > 120 cm/sec. The diagnosis of increased intracranial pressure (ICP) includes non-invasive methods like neurologic examination, CT or magnetic resonance (MR). Invasive methods include external ventricular drainage with measurement of ICP greater than 20 mmHg for more than 5 to 10 minutes.33 We also measured the midline shift measurement by drawing a vertical line from anterior of the falx cerebri to posterior insertion. A perpendicular line was drawn to the vertical line and measured from the line to the septum pellucidum, at the level of the foramen of Monro. To minimize measurement errors, the measurements were performed by two board-certified neurosurgeons who were blinded to treatment assignment and the mean value was documented. Hemorrhage and PHE were volumetrically measured with CT using 1.5 to 5 mm-thick slices; the slices were processed using software developed in-house (AsanJ, based on a plug-in package for ImageJ [https://rsbweb.nih.gov/ij]).

The fundamental management of SAH was performed according to standard guidelines.31 Surgical decisions including treatment modality and techniques were at the discretion of the attending neurosurgeons or neurointerventionists. Microsurgery or endovascular treatments were performed within 24 hours of admission. Fentanyl (0.5–5 µg/kg/hour) or remifentanil (0.025–0.2 µg/kg/min) IV was administered in cases where continuous analgesics were needed. CTA or MRA was performed whenever vasospasm was suspected. The medical treatment of vasospasm was in accordance with SAH guidelines.31 Patients with vasospasm refractory to medical management were treated with angioplasty using infusion of intra-arterial vasodilating agents like nicardipine. The prophylactic anticoagulants were not administered in the study enrolled patients. Instead, intermittent pneumatic compression and bedside physical therapy were applied to all patients with risk of thrombosis. Discharge from ICU and transfer to general ward is indicated if the patient’s vital functions are stable without the intensive care support and no longer require monitoring or treatment.

Among various antioxidants, the effect of acetylcysteine and selenium in aSAH patients has not been well studied. Also there were no studies focusing on the same endpoints as the current study to date. Accordingly, we estimated the sample size from past studies which have included other antioxidants targeting ICH patients. Based on an expected effect size of antioxidants in aSAH patients (0.6), assuming 5% lost to follow-up, 106 subjects need to be randomized 1:1 antioxidants vs. placebo afford 85% power to demonstrate with a 2-sided α of 0.05. The expected effect size of 0.6 was derived from the study results administrating statin (antioxidant) to ICH patients. Cohen’s D effect size calculation was used to calculate the expected effect size.

Categorical data were analyzed using the Pearson χ² test or Fisher’s exact test whereas Student’s t-test was used to compare quantitative data. All values are presented as mean with standard deviation or median (interquartile range 25th to 75th percentile). Univariate analysis was performed with clinical factors of interest. Subsequent stepwise multivariate logistic regression analysis was performed to assess the association between the antioxidant and these factors. Backward elimination (95% confidence interval, CI) method was used to determine the final model, in which all remaining factors had a P < 0.15. All statistical analyses were performed with the use of SPSS (version 21; SPSS Inc., Chicago, IL, USA) and SAS 9.4 (SAS Institute, Cary, NC, USA). Statistical significance was considered when P value < 0.05.

This prospective, multi-center, singled-blind, randomized study was developed by Korean neuro-ICUs with approval from the Institutional Review Board (Asan Medical Center, 2019-1337). Written informed consent was obtained from all participants, families, or their legal representatives before enrollment in this study.

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. We confirm that our clinical trial was registered and approved according to the clinical research information service. This is a randomized clinical trial, registered at the Clinical Research Information Service (CRIS, http://cris.nih.go.kr, unique identifier: KCT0004628).

We prospectively screened 293 SAH patients between January 2017 and October 2019 from three different tertiary medical centers. Among these patients, 187 were excluded as per the inclusion and exclusion criteria (n = 171), declining to participate in the study (n = 14) or death prior to assignment (n = 2). Accordingly, 106 patients were included in the assignment of antioxidants, but 3 patients withdrew consent before commencing intervention. Finally, 103 aSAH patients were included in the present study. Among these patients, 53 were assigned to the antioxidant group and 50 to the non-antioxidant (placebo) group (Fig. 2).

There were no significant differences in admission factors between the antioxidant and non-antioxidant groups; age (57.20 ± 16.22 vs. 59.01 ± 13.22 years, P = 0.511), male sex (21 vs. 24, P = 0.843) and Hunt & Hess grade (grade 1, 26% vs. 18.9%; grade 2, 30% vs. 34%; grade 3, 26% vs. 24.5%; grade 4, 14% vs. 22.6%; and grade 5, 4% vs. 0%, P = 0.476). Radiologic assessments of ruptured aneurysm locations (internal carotid artery, 36% vs. 32.1%; anterior cerebral artery, 24% vs. 28.3%; middle cerebral artery, 18% vs. 22.6%; posterior cerebral artery, 16% vs. 9.4%; anterior inferior cerebellar artery, posterior inferior cerebellar artery and basilar artery, 6% vs. 7.5%, P = 0.149) and modified Fisher grade (grade 1, 2, and 3, 66% vs. 64.23%; grade 4, 34% vs. 35.8%; P = 0.846) showed no differences. There was also no significant difference in the type of operations performed between the groups (clipping, 52% vs. 52.8%; bypass and clipping, 4% vs. 5.7%; bypass and trapping, 6% vs. 3.8%; decompressive craniectomy, 12% vs. 15.1; coil embolization, 33% vs. 30.2%; bypass and coil embolization, 2% vs. 3.8%; and trapping 4% vs. 3.8%, P = 0.132). No significant differences were observed in admission neurologic score (GCS 11 [7–14] vs. 10 [7.5–14], P = 0.194 and mRS 4 [3–5] vs. 5 [3–5], P = 0.237), agitation (RASS −1 [−3–0] vs. −2 [−4–0], P = 0.382). The comparison of SAH managements during ICU admission, such as continuous analgesics (72% vs. 69.8%, P = 0.984) and osmotic agents (mannitol, 38% vs. 39.6%; hypertonic saline, 0% vs. 1.9%; and both 2% vs. 1.9%, P = 0.779) showed no differences. The results are summarized in Table 1.

The radiological impact of antioxidants in reducing PHE were only focused on modified Fisher grade 4 aSAH patients as these patients have localized ICH. CT and CTA scans upon admission showed no differences in the volume of hematoma and PHE causing mid-line shifting and vasospasm. Administration of antioxidants showed no beneficial effects of PHE reduction and midline shifting (Fig. 3). No significant differences in incidence of radiological vasospasm were observed between the two groups at the 1-week (11 patients [22%] vs. 9 patients [17%], P = 0.525) or 2-week (15 patients [30%] vs. 14 patients [26.4%], P = 0.690) radiologic evaluation. The incidence of angioplasty for vasospasm (12% vs. 11.3%, P = 0.916) and diagnosis of hydrocephalus on 2-week CT or MR (26% vs. 28.3%, P = 0.829) showed no differences too. The results are summarized in Table 2.

There were no significant differences between the antioxidant and non-antioxidant groups on time to reach target RASS (5.4 ± 3.8 vs. 5.9 ± 4.1 hours, P = 0.122) and the need for tracheostomy (5 [10%] vs. 7 [13.2%], P = 0.284). However, significant differences were observed in total ICU stay (11.6, 95% CI, 9.7–13.4 vs. 8.9, 95% CI, 7.9–10.7 days, P = 0.044) despite no differences being observed in the total hospital stay (24.2, 95% CI, 21.3–27 vs. 22.4, 95% CI, 19.6–26.1 days, P = 0.107). The 30-day neurologic outcomes using mRS were not different between these 2 groups (2.8, 95% CI, 2.1–3.4 vs. 2.4, 95% CI, 1.9–3.1, P = 0.088) (Fig. 4). We further analyzed these factors using multivariate analysis to show the clinical impact of antioxidants. The significant improvements were observed in total ICU stays (11.2, 95% CI, 9.7–14.5 vs. 8.3, 95% CI, 6.2–10.2 days, P = 0.008) but fail to show in total hospital stay (23.2, 95% CI, 20.9–26.5 vs. 21.9, 95% CI, 18.9–24.2 days, P = 0.123) or 30-day mRS (2.6, 95% CI, 1.9–3.3 vs. 2.2, 95% CI, 1.5–2.9, P = 0.105). The results are summarized in Table 3.

A total of 19 SAEs were reported in 17 patients and none of them were potentially associated to our antioxidant drugs. The most common SAEs were pneumonia or ARDS (non-antioxidants, n = 6 vs. antioxidants, n = 4, P = 0.450) followed by acute kidney injury (non-antioxidants, n = 1 vs. antioxidants, n = 3, P = 0.341), venous thrombosis events (non-antioxidants, n = 2 vs. antioxidants, n = 1, P = 0.529), heart injury (non-antioxidants, n = 1 vs. antioxidants, n = 1, P = 0.967) and sepsis or septic shock (non-antioxidants, n = 1 vs. antioxidants, n = 0, P = 0.306). No significant differences in the incidence of SAE were observed between the two groups. The results are summarized in Table 4.

The serum selenium levels on admission were within normal limits for both groups (non-antioxidant, 8.4 ± 3.4 vs. antioxidant, 7.9 ± 2.9 µg/dL, P = 0.258). However, the antioxidant group showed a significant increase in serum selenium levels on day 7 (9.2 ± 4.5 vs. 13.5 ± 4.2 µg/dL, P < 0.01) and day 14 (8.7 ± 4.5 vs. 19.6 ± 3.8 µg/dL, P < 0.01) after selenium administration. There were 9 patients whom exceeded the upper limit of serum selenium level after the drug administration (non-antioxidant [n = 5, 10%] vs. antioxidant [n = 7, 13.2%], P = 0.743). The administration was paused for 1 time in all these patients and resumed in the next schedule. The results are summarized in Table 5.