Inammatory factors are associated with early neurological deterioration in acute ischemic stroke patients receiving endovascular therapy-The AISRNA study

Background: This study aimed to explore several peripheral blood-based markers related to inammatory response in a total of 85 patients with acute ischemic stroke (AIS) caused by large artery occlusion in the anterior circulation receiving endovascular therapy (EVT) from the AISRNA study regarding the association between inammatory factors and early neurological deterioration (END), and investigated whether their time course correlated with END after EVT. Methods: We collected baseline characteristics of 85 AIS patients participating in an observational acute stroke cohort: the AISRNA study. The following inammatory factors were measured in these participants: interleukin-2 [IL-2], IL-4, IL-6, IL-10, tumor necrosis factor-α [TNF-α], and interferon-γ [IFN-γ]. The National Institute of Health Stroke Scale score increase of ≥ 4 within 24 hours after EVT dened as END. Results: IL-6 and IL-10 were higher in patients with END compared to those with non-END (P<0.01), and they were also associated with risk factors of END after EVT. Furthermore, we found that the area under curves (AUCs) of IL-6 and IL-10 for predicting END were 0.791 (0.689-0.871), and 0.564 (0.452-0.671), respectively. Adjusting for age, sex, and atrial brillation, the odds ratios (ORs; 95% condence interval) for incident END for IL-6 and IL-10 were 1.83 (1.08-6.36) and 1.15 (1.02-1.30), respectively. Additionally, we found signicant changes over time in the expression levels of IL-4, IL-6, and IL-10 in patients undergoing END compared with non-END (P<0.05). Conclusions: IL-6 and IL-10 levels on admission are signicantly associated with END after EVT, and time course of IL-4, IL-6, and IL-10 is correlated with stroke progression. Further study of molecular mechanisms on peripheral immunomodulation in AIS would be helpful. interquartile range; Infarction NIHSS, National Institute of Health Stroke Scale; LAA, large artery atherosclerosis; TC, total cholesterol; TG, triglyceride; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein.


Introduction
Stroke is commonly considered as a major cause for adult disability and mortality. Although the early advent of endovascular therapy (EVT) has obviously improved clinical outcome of acute ischemic stroke (AIS), over 50 percent of patients still suffer from disabilities and de cits, which may be the result of neurological and medical complications (1). Precise prediction of clinical outcome remains challenging during the acute phase of ischemic stroke (IS) after EVT. While our previous studies have found that demographic and several clinical characteristics are associated with prognosis after acute ischemic stroke (2)(3)(4), the accuracy of prediction remains limited, especially during the acute phase of IS (5). Therefore, development of precise scores to predict prognosis in the acute stage may bene t from the individual biomarkers.
The correlation between stroke and acute in ammatory response can be found in various stages of acute infection. Accumulating evidence has indicated that stroke induces a rapid immunodepression through the autonomic nervous system (6,7). Immune factors including cytokines are indicative to the strokeassociated infection and associated with clinical outcome after AIS (8,9). However, the link between in ammatory factors and stroke progression remains unclear in patients receiving EVT.
In the present study, we aimed to explore the association between a range of in ammation-related biomarkers (interleukin-2 [IL-2], IL-4, IL-6, IL-10, tumor necrosis factor-α [TNF-α], and interferon-γ [IFN-γ]) and early neurological deterioration (END) after stroke caused by large artery occlusion in the anterior circulation. We enrolled of a total of 85 patients receiving EVT from the AISRNA study to investigate the link between in ammatory factors and END, and observed that high levels of IL-6 and IL-10 were associated with END. Additionally, time course of IL-4, IL-6, and IL-10 is correlated with stroke progression after EVT.

Study population
Our study protocol was approved by the Nanjing Medical University Ethics Committee and followed the Declaration of Helsinki. We enrolled 85 AIS patients caused by large artery occlusion in the anterior circulation receiving EVT in the present study. Firstly, a total of 85 AIS patients receiving EVT were prospectively collected from the AISRNA study (www.clinicaltrials.gov, NCT04175691) to investigate the association of in ammatory factors on admission and their dynamic changes with END for 7 days. Their clinical characteristics are summarized in Table 1. All patients were from Nanjing First Hospital, Nanjing Medical University which is a stroke center a liated to China Stroke Association. Eligible patients were collected in the present study if they followed these inclusion criteria: (1) AIS patients receiving EVT; (2) the anterior circulation occlusion; (3) followed standard in-house procedures (4); and exclusion criteria: (1) preexisting dysphagia; (2) intracranial hemorrhage; (3) infection on admission; (4) antibiotic or immunodepression medical therapy within 4 weeks; (5) age less than 18 years; (6) history of a malignant tumor; (7) died within 7 days.

Clinical data
Demographics, medical history, stroke severity on admission and 24 hours, stroke etiology, laboratory parameters, modi ed Thrombolysis in Cerebral Infarction Score [mTICI], and time of onset to door, groin puncture, and reperfusion. Several data de nitions were used in the present study: the National Institute of Health Stroke Scale (NIHSS) was used to assess stroke severity; the delta NIHSS scale (delta NIHSS 0-24h) = NIHSS on admission -NIHSS after 24 hours; early neurological deterioration (END) was de ned as ΔNIHSS ≥4 points (10); stroke etiology was determined according to the Trial of Org 10 172 in acute stroke treatment (TOAST) criteria (11).

Statistical analysis
Categorical variables are expressed as frequency and percentage. Continuous variables of an abnormal distribution are expressed as medians (interquartile range [IQR]), and normally distributed variables as mean ± standard deviation (SD). The t-test, one-way ANOVA and Mann-Whitney U-tests were used to analyze continuous variables if necessary. Bivariate correlation between delta NIHSS score and in ammatory factors was analyzed by the Spearman correlation. Variables from signi cant factors of the univariable logistic regression analysis were considered in the multivariate logistic regression model to obtain independent factors. Discriminatory capacities of in ammatory factors was assessed by receiver operating characteristic (ROC) curve analyses and area under the curve (AUC). The signi cance threshold was set at less than 0.05.
A total of 10 (11.8%) patients underwent END after EVT. The association between in ammatory factors on admission and incidence of END is shown in Table 1. The NIHSS on admission was no signi cant difference in both groups (P = 0.378). However, compared with non-END, NIHSS was signi cantly higher in patients with END after 24 hours (P < 0.001). We found that END was associated with increased IL-6 and IL-10 levels as well as higher proportion of atrial brillation (P < 0.05). As the delta NIHSS was considered as END, we performed a correlation analysis to investigate the correlation between in ammatory factors and delta NIHSS 0-24 h. The results showed that the delta NIHSS was correlated with the expression of IL-6 (r = 0.260, P = 0.016) and IL-10 (r=-0.238, P = 0.028) ( Fig. 2B and 2C). Additionally, we performed a ROC curve analysis to explore their predictive powers. We observed that the AUCs of IL-6 and IL-10 for predicting END were 0.791 (0.689-0.871), and 0.564 (0.452-0.671), respectively (Fig. 3), revealing that IL-6 outperformed IL-10 in predicting END (P < 0.001).
Given that association of in ammatory factors with END, we conducted a binary logistic regression to analyze the impact of in ammatory factors on END. The results of the univariate analyses showed that IL-6 and IL-10 levels were prognostic factors for END, and the ndings remained stable after adjustment ( Table 2).
To further study the clinical utility of in ammatory factors after EVT, we analyzed time courses for in ammatory factors within 7 days for END and non-END (Fig. 4). Among these patients, there were 85 samples for day 1 (on admission), 58 for day 2, and 28 for day 3 and 7. We found signi cant changes over time in the expression levels of IL-4, IL-6, and IL-10 in patients undergoing END compared with non-END (P < 0.05), and IL-6 levels were obviously increased from days 1 to 7 after EVT (Fig. 4B, P < 0.001). Furthermore, we observed IL-4 and IL-10 peak levels at 2 days, and then rapidly decreased at 3 days.
Additionally, patients with END had decreased IL-10 levels compared with those with non-END at 7 days ( Fig. 4C, P < 0.01).

Discussion
This study of EVT participants from the AISRNA study showed a strong association of IL-6 and IL-10 levels on admission with risk of END. Our study suggested that patients with increased IL-6 and IL-10 levels had higher risks of developing post-EVT END. By contrast, there was no correlation of baseline levels of IL-2, Il-4, INF-γ, and TNF-α with END. The predictive power of IL-6 for END was superior to that of other in ammatory factors. Additionally, IL-4, IL-6, and IL-10 levels presented a marked rising trend in patients with END.
In ammation is a hallmark of stroke etiology and progression. Post-stroke in ammation is accounted as a requisite pathological process involved in ischemic brain injury (12). A series of detrimental complications occur and the blood-brain barrier (BBB), damaged after the initial brain injury, crossed by activated peripheral immune cells including monocytes and T-cells (13). Furthermore, post-stroke in ammatory response is associated with stroke severity on admission as determined by NIHSS (14). Additionally, patients who underwent END were more susceptible to have a poor functional outcome after 90 days (15). However, the association between post-stroke in ammatory response and END remains uncertain. Therefore, we performed a prospective study from the AISRNA study to further investigate the in uence of in ammatory factors on END after stroke.
The biological function of IL-6 in AIS remains unclear. Mounting evidence have demonstrated that IL-6 levels are increased in peripheral blood samples during the rst 7 days after stroke onset. For example, increased IL-6 was associated with the infarct size and stroke severity on admission (16) as well as risk of incident stroke (17), although a study reported the opposite that early IL-6 levels, as a neuroprotective factor, was inversely correlated with lesion size and functional outcome (18). Our ndings also suggested that increased IL-6 was associated with risk of END after EVT. However, there is a less agreement on the time point of peak IL-6 levels. Some studies reported IL-6 peak levels at 3 day (19,20), which are similar to our ndings. Other describe high levels of IL-6 with ranged from a few hours till one day or rst week after stroke (18, 21, 22).
We also reported positive correlation between IL-10 and END after EVT. Previous studies have suggested IL-10 as a marker for incident stroke (23,24). IL-10 is a major anti-in ammatory cytokine to regulate the immune response by suppressing pro-in ammatory cytokine expression. The in uence of IL-10 on functional outcome and its time course after EVT remains uncertain. Several investigators reported that IL-10 may serve a neuroprotective role and predict clinical outcome after stroke (25,26). However, our previous studies from the experimental rats and human stroke patients observed that IL-10 was positively associated with stroke risk (27,28). In the current study, increased IL-10 levels on admission were associated with END after EVT. Whereas we observed IL-10 peak levels at 2 days, and patients with END had decreased IL-10 levels compared with those with non-END at 7 days. This phenomenon may be due to a stress response of IL-10 regarding END which lead high levels of IL-10, but its levels were subsequently decreased after 2 days. Therefore, IL-10 may be involved in the process of END, and its molecular mechanisms remain to be explored in the further study.
IL-4, an anti-in ammatory cytokine, can drive Th2 cell differentiation which plays bene cial roles in inhibiting post-stroke in ammation, repairing damaged brain tissues and inducing neurotrophic factors in astrocytes (29,30). A previous study has demonstrated that IL-4 was signi cantly correlated with stroke severity and functional outcome in AIS patients (31). However, our results showed no association of IL-4 with END after EVT, but IL-4 levels were increased in patients with END at 2 days and then rapidly decreased at 3 days. At present, we are also investigating its molecular mechanisms regarding dynamic change of IL-10 in stroke progression.
Several limitations should be acknowledged in the study. First, this is a small number of patients in a single center, but we are seeking for sub-centers to complete the AISRNA study. Second, we did not analyze the association between initial infarct burden and END after EVT. Finally, molecular mechanisms regarding dynamic changes of these in ammatory factors should be further explored in patients undergoing EVT.

Conclusions
In summary, this study illustrates the in uence of in ammatory factors on END and their time course after EVT among AIS patients caused by large artery occlusion in the anterior circulation. We found that serum concentration of IL-6 and IL-10 on admission is obviously associated with END after EVT, and time course of these factors is correlated with stroke progression. Thus, peripheral immunomodulation in AIS remains to be further studied to provide a new pharmacological approach with stroke therapy.

Declarations
Ethics approval and consent to participate This study protocol was approved by the ethics committee of Nanjing First Hospital, Nanjing Medical University and followed the tenets of the Declaration of Helsinki. Written informed consent was obtained from all individuals or their family members. This study design is based on the stroke registry of Nanjing First Hospital, Nanjing Medical University, a national advanced stroke center a liated with Stroke Prevention Project, National Health Commission.

Consent for publication
Not applicable.

Availability of data and materials
All data supporting our results are available from the corresponding authors upon reasonable.

Declaration of interests
The authors declare that they have no competing interests.   Figure 1 Flowchart of the study patients to illustrate study screening, recruitment, and follow-up. END, early neurological deterioration.

Figure 4
Time course of in ammatory factors after stroke onset (days 1 to 7). Signi cant changes over time were observed in the expression levels of IL-4, IL-6, and IL-10 in patients undergoing END compared with non-END (P<0.05). IL-6 levels were obviously increased from days 1 to 7 after EVT (B). IL-4 (A) and IL-10 (C) peak levels were at 2 days, and then rapidly decreased at 3 days. END, early neurological deterioration. *P<0.05, **P<0.01, ***P<0.001.