Total cholesterol affects the outcome in anterior cerebral artery-occluded acute ischemic stroke patients treated with thrombolysis

Object In this study, we investigated whether certain types of lipid profiles are major contributors to disease outcomes. At two stroke medical centers, among 13,285 hospitalized patients treated with thrombolysis, thrombectomy, or conventional care, two hundred and seventy-six anterior cerebral artery-occluded acute ischemic stroke (AIS) patients were screened. We examined the plasma lipid profiles by using the cutoff values from a receiver operating characteristic (ROC) curve. A multivariate logistic regression or Fisher’s exact test was used to compare the outcome and risk events. The modified Rankin scale (mRS) score was used to assess the major clinical outcome of the patients 3 months after disease onset. Mortality and symptomatic intracranial hemorrhage (sICH) were both evaluated as risk factors. The disease outcome was examined by analyzing symptom improvement at discharge.


KEYWORDS
acute ischemic stroke; thrombolysis; total cholesterol; atherosclerosis Abstract Object In this study, we investigated whether certain types of lipid profiles are major contributors to disease outcomes.

Methods
At two stroke medical centers, among 13,285 hospitalized patients treated with thrombolysis, thrombectomy, or conventional care, two hundred and seventy-six anterior cerebral artery-occluded acute ischemic stroke (AIS) patients were screened. We examined the plasma lipid profiles by using the cutoff values from a receiver operating characteristic (ROC) curve. A multivariate logistic regression or Fisher's exact test was used to compare the outcome and risk events. The modified Rankin scale (mRS) score was used to assess the major clinical outcome of the patients 3 months after disease onset.
Mortality and symptomatic intracranial hemorrhage (sICH) were both evaluated as risk factors. The disease outcome was examined by analyzing symptom improvement at discharge.

Conclusion
In anterior cerebral artery-occluded AIS patients, the TC level is a highly promising screening factor for predicting the outcome of IV thrombolysis.

Background
Atherosclerotic cardiovascular disease and its clinical manifestations, such as ischemic cerebrovascular disease and coronary artery disease, are the leading cause of morbidity and mortality worldwide 1 2, 3 . In most epidemiological cohort studies, the total cholesterol (TC) levels are well known to be associated with the cause of stroke. An increased risk of intracerebral hemorrhage (ICH) is closely associated with lower cholesterol levels 4 .
Patients with higher TC levels are susceptible to ischemic stroke 5-10 . Therefore, determining whether certain types of plasma lipid profiles play critical roles in causing stroke is important. Lower HDL-C and TG levels have been used as two important parameters to independently predict a better survival rate after intravenous (IV) thrombolysis treatment in a retrospective analysis 11 , and it has been well documented that patients can greatly benefit from this treatment. Lower low-density lipoprotein cholesterol (LDL-C) levels were found to be associated with ICH, while high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels do not differ between stroke patients with or without ICH 12 . It is apparent that different types of biological lipoproteins are associated with certain disease types and their prognosis or consequences. Over the past decade, few studies have focused on the association between TC levels and prognosis in stroke patients. Thus, identifying the association between an individual's plasma lipid profile and prognosis in AIS is critical.
In this study, we evaluated the association between the lipid profiles and prognosis in AIS patients. We analyzed the relationship between cholesterol levels and prognosis following thrombolysis. Subsequently, we compared the effect of cholesterol on three common AIS management strategies (thrombolysis, thrombectomy and conventional treatment).
Importantly, we found that the lipid profiles presented in patients' plasma can be used as indicators to predict the overall outcome of severe anterior cerebral artery-occluded AIS  Figure 1.
According to the baseline NIHSS score, which is a clinical measure of neurologic deficit with a range of 0 (no deficit) to 42 (maximum possible deficit), patients with a score of 10 or more, representing a greater than 80% likelihood of a major arterial occlusion 13 , were identified. A stroke neurologist clinically assessed all patients upon admission. Cranial computed tomography (CT) was obtained prior to IV recombinant tissue plasminogen activator (rt-PA) treatment to rule out intracranial hemorrhage.
IV rt-PA thrombolysis treatment was initiated within 4.5 hours of the onset of stroke symptoms, and mechanical thrombectomy treatment was initiated within 6 hours as we previously reported [14][15][16] . The conventional care group consisted of patients who were subject to high risks during thrombolysis or thrombectomy or whose families did not consent to treatment. The time between onset and admission was less than 6 hours.

Patient treatment
The main inclusion criteria were as follows: 1. an NIHSS score ≥ 10 at admission; 2.
imaging evidence revealing that the occlusion site was the anterior cerebral artery; and 3.
analysis of the full lipid profile (including TG, TC, HDL-C and LDL-C) within 24 hours of stroke onset. The exclusion criteria included uncontrolled hypertension, severe sensitivity to radiographic contrast agents, and CT or magnetic resonance imaging (MRI) evidence of intracranial hemorrhage.
The patients in the thrombectomy group were treated as previously described [14][15][16] . The technical details of the endovascular procedure have also been described in a previous study. Upon admission, a stroke neurologist examined all patients; cranial CT or multimodal MR angiography was performed on patients prior to each intervention to confirm the diagnosis of large-vessel occlusion and rule out intracranial hemorrhage. All interventional treatments were initiated within the first 6 hours of stroke onset. For the thrombolysis treatment, we followed the American Heart Association (AHA) guidelines 17 as described in a previous study 16  and MR images are very useful tools for evaluating the infarct lesion in the brain. The Alberta Stroke Program Early CT Score (ASPECTS) was used as a grading system to standardize the quantification of the CT images 22 . As the ASPECTS criteria were not included in the treatment guidelines of our two centers, ASPECTS was not measured immediately before the rt-PA treatment and did not affect the choice of management strategy. ASPECTS was evaluated by two neurosurgeons at the initial stage of this retrospective study (CJ.G., Y.L., and Y.P. contributed to this work). The stroke mechanism was viewed according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification. The subtypes of AIS were defined at the 3-month follow-up as follows: cardioembolic, large-vessel atherosclerosis, other (uncommon etiologies), or undetermined 23 . When no etiology was found or two diagnoses were possible, the etiology was classified as undetermined. The outcome at 3 months was evaluated by the modified Rankin scale (mRS) score either at a face-to-face visit or by telephone. Favorable outcomes were defined as a score of 0 to 2, and unfavorable outcomes were defined as a score of 3 to 6.
First, the mRS score at 3 months (the major outcome variable) was evaluated in the patients in the thrombolysis group with TC ≤ 5.07 mmol/L or > 5.07 mmol/L. Then, the mRS score was compared with that in the thrombectomy group, the thrombolysis group, and the conventional care group. We also checked for symptom improvement after different management techniques. To evaluate the degree to which the symptoms improved after treatment, we compared the clinical improvement (discharge NIHSS scoreadmission NIHSS score ≥ 4) and the marked improvement (discharge NIHSS scoreadmission NIHSS score ≥ 10) rates of the patients among the treatment groups.
Additionally, the difference in sICH and mortality risk between the thrombectomy (or thrombolysis) and conventional care groups was evaluated. The following data were also collected: age, gender, stroke reason, time between onset and needle, blood pressure at admission, plasma glucose and lipid profiles, medical history, documented occlusion site, and mRS score at 3 months.

Statistical analysis
The baselines and main outcomes are described as the means and standard deviations; the qualitative data are described as numbers and percentages. The lipid profiles were stratified using the cutoff values from the receiver operating characteristic (ROC) curve.
For comparisons between different therapies, a Chi-square test or 2-sided Fisher's exact test was used (significance was set as p < 0.05). The independent prognostic value of the lipid profiles, the associations among the different management options, and symptomatic improvement or some other risk events were analyzed by a bivariate logistic regression analysis. All statistical analyses were performed using SAS software (version 9.4, SAS Institute).

Prognostic factors of the clinical outcome in thrombolysis patients
Because blood lipids are reportedly inflammatory mediators and have been considered high risk factors of stroke, we examined the lipid profiles in plasma by using the cutoff values from the ROC curve. In this study, for the severe anterior cerebral artery-occluded AIS patients treated with thrombolysis, the cutoff values of TG, TC and LDL-C were 1.65 mmol/L (sensitivity 71.4%, specificity 87.5%), 5.07 mmol/L (sensitivity 83.3%, specificity 58.5%), and 3.01 mmol/L (sensitivity 63.5%, specificity 81.2%), respectively.
To determine whether the lipid profiles are predictors of the disease outcome, a univariate logistical regression was conducted. As shown in Table 1 (Table 1).

Population baseline
We also sought to determine whether the lipid profiles affect the prognosis of AIS patients   Table  2.

Clinical outcome
To determine whether TC affected the overall outcome, the mRS score of the patients in the different treatment groups at 3 months was analyzed based on the TC cutoff value. In addition to our previous analysis, a TC level > 5.07 mmol/L corresponded to a 3.55-fold

Mortality and sICH
The mortality rate of the patients with a TC level > 5.07 mmol/L was 13.30% (4 of 30 patients) in the thrombolysis group and did not show a significant advantage compared with that in the control group (OR 0.60, 95% CI 0.08-4.32  Table   5).
The symptoms of intracranial hemorrhage during therapy were considered risk factors for a disability outcome. In total, 6 (6.30%) and 9 (11.54%) patients suffered from sICH within 24 hours of treatment in the thrombolysis and thrombectomy groups, respectively, as shown in Supplementary Table 3. Two sICH (1.85%) occurred 24 hours after conventional care. Considering the TC cutoff value (5.07 mmol/L), there were no significant differences between the thrombolysis and thrombectomy groups as shown in Table 6.

Discussion
The type of lipid profile and lipid accumulation at the location of cerebral large-artery occlusion have been reported to be critical factors of disease outcome. The efficacy of IV thrombolysis in large-artery occluded AIS patients is still controversial. Despite the extensive studies that have been conducted, the association between lipid profiles and AIS prognosis is highly controversial and remains unclear 4, 8, 11, 12, 24-26 . Thus, excluding patients with severe illnesses, we questioned whether other factors interfere with the efficiency of IV thrombolysis. As a result, our data demonstrated that the TC level was an independent prognostic factor for IV thrombolysis. Higher TC levels (> 5.07 mmol/L) independently predicted poor outcomes after thrombolysis treatment in severe anterior cerebral artery-occluded AIS patients.
Furthermore, our study aimed to identify the association between the patterns of plasma lipids and the outcome and prognosis of AIS and determine whether the best treatment strategy is either thrombolysis or mechanical thrombectomy. By performing a ROC curve analysis, we determined the relationships between the outcome of AIS and the plasma lipid profiles. More importantly, we provide a relatively definitive boundary that can help predict the outcome of disease based on plasma lipid levels. The threshold TC level of 5.07 mmol/L can be used as an indicator to predict disease outcome 3 months after disease onset among patients managed by IV thrombolysis.
A retrospective analysis of 1066 AIS patients reported that there was no association between lipid profiles and 3-month mRS scores or sICH 27 . Additionally, other studies have shown that lower plasma TC, TG and HDL-C levels can each independently predict a poor 3-month outcome 26 . In contrast, as described elsewhere, a higher HDL-C level has also been shown to be associated with a favorable outcome at 3 months 11 . However, unexpectedly, higher HDL-C and TG levels were independently correlated with increased mortality in the same study 11 . Our data clearly show that there is a close association between TC, but not TG and HDL-C, and the outcome of disease. In agreement with other studies, we did not observe an obvious association between the lipid profiles and sICH.
Because the administration of statin can lower cholesterol levels, our data support the notion that lowering cholesterol levels does not affect the sICH risk in patients treated with IV thrombolysis.
The mechanism underlying the association between higher TC levels and poor 3-month outcome remains unclear. High plasma TC levels have been shown to be strongly associated with atherosclerosis stroke in large sample cohort studies performed by other researchers 9, 10 . The unfavorable outcome observed in AIS patients receiving IV thrombolysis might be caused by large-vessel atherosclerosis 28-30 , which is closely associated with high levels of TC.
It is a general consensus that the poor artery recanalization of thrombolysis in stroke patients caused by atherosclerosis might lead to a poor prognosis 31 . The clinical outcome of severe AIS patients (NIHSSS ≥ 10) treated by thrombectomy does not significantly differ between patients divided by the TC threshold (> 5.07 mmol/L and ≤ 5.07 mmol/L) (Table 3). To further confirm whether the TC levels influence the clinical outcome after thrombectomy treatment in severe anterior cerebral artery-occluded AIS patients, we replotted the ROC curve based on another TC cutoff value obtained through the thrombectomy database. The subsequent multivariable logistic regression including the new TC cutoff value still did not reveal a significant difference as shown in Supplementary   Table 3. Therefore, the efficacy of thrombectomy might not be linked to cholesterol levels given the excellent recanalization rate of the thrombectomy procedure. However, this result seems to contradict the lack of association between atherosclerosis and outcome in our logistic regression model. Thus, we speculate that the etiological classification is a subjective measurement that might represent some deviation. The artificial etiological classification might not truly reflect the real condition of AIS patients. Our data suggest that the TC level could be a much more informative parameter that accurately reflects the atherosclerosis status of patients. Thus, in severe anterior cerebral artery-occluded AIS patients, the TC level might be a useful screening parameter for IV thrombolysis.
Finally, as stated in the materials and methods section, the included patient samples might be insufficient and could limit the strength of our study. However, the data collected are significant according to the ROC curve analysis. Nonetheless, further work is needed for prospective clinical trial verification.

Conclusions
In anterior cerebral artery-occluded AIS (NIHSS ≥ 10) patients, a TC level greater than 5.07 mmol/L can be considered a threshold for predicting a poor outcome following IV thrombolysis treatment, and if the TC level of an AIS patient is above this threshold, it is recommended that the physician choose an appropriate therapeutic strategy, such as mechanical thrombectomy. The determination of the TC levels is a useful screening method for the prediction of the outcome of IV thrombolysis.

Declarations
Meaney from the University of Utah, Salt Lake City, United States of America for helping us thoroughly review the manuscript.    * TC = total cholesterol. † mRS score = modified Rankin scale score. ‡ This analysis was performed by using a logistic regression. * TC = total cholesterol †This analysis was performed by using a logistic regression.