DOI: https://doi.org/10.21203/rs.3.rs-2116708/v1
Decreased eosinophil level was associated with poor outcome after mechanical thrombectomy in patients with acute ischemic stroke (AIS), but pathogenesis of this association is elusive. We aimed to assess the mediation effect of intracranial bleeding complications on the aforementioned association.
A total of three hundred and twenty-eight consecutive AIS patients experiencing mechanical thrombectomy between May 2017 and March 2021 were analyzed. Hemorrhagic transformation (HT) were categorized as symptomatic intracranial hemorrhage (sICH) and parenchymal hematoma (PH) according to previously published criteria. Regression analysis was used to assess the effect of eosinophils on HT, and its effect on poor outcome. Mediation analysis was utilized to assess the proportion of total effect by HT on the association between eosinophils and poor outcome.
Multivariater analysis revealed that eosinophils was independently associated with sICH after adjusting for potential confounders (odds ratio, 0.00; 95% CI, 0.00–0.01; P = 0.0141), which is consistent with the result of eosinophils (dichotomous) as a categorical variable (odds ratio, 0.22; 95% CI, 0.11–0.46; P < 0.0001). And the risk of PH in higher eosinophils was 0.36 fold higher than in patients in the low eosinophil group (OR, 0.36, 95% CI, 0.19–0.67; P = 0.0013). Eosinophils was negative associated with poor outcome (odds ratio, 0.00; 95% CI, 0.00–0.02; P = 0.0021). And mediation analysis found that sICH partially mediated the negative relationship between eosinophils and poor outcome (indirect effect=-0.1896; 95%CI: -0.3654 – -0.03, P < 0.001); however, PH did not mediate the association between eosinophils and poor outcome (P = 0.12).
This study showed an important effect of sICH on the association between eosinophils and poor outcome.
Reduced eosinophil levels are associated with poor prognosis after mechanical thrombectomy in patients with acute ischemic stroke (AIS), but the pathogenesis of this association is unclear [1]. In contrast, our previous study found that lower eosinophil levels were associated with higher stroke associated pneumonia (SAP) and that SAP played an important mediating role in the association between eosinophilia and poor prognosis. Furthermore, this study found a significant association between eosinophils and poor outcome independent of SAP in a multivariate analysis [1]. These results suggest that eosinophils may contribute to poor prognosis through other pathways besides through SAP, such as cerebral ischemia-reperfusion injury and subsequent intracranial hemorrhagic complications.
Eosinophils can secrete a variety of chemokines and vascular endothelial growth factors [2–4]. Chemokines secreted by eosinophils can induce activation of M2 phenotype microglia and are neuroprotective by promoting the resolution of inflammation. In contrast, vascular endothelial growth factor may be neuroprotective by regulating angiogenesis [2–4]. Furthermore, it is possible that the decrease in eosinophils leads to more cerebral ischemia-reperfusion injury, which in turn leads to hemorrhagic transformation (HT) and subsequent poor consequences. In this view, it is reasonable to assume that an increase in HT may be in the path of association between eosinophilia and poor outcomes.
In the present study, we aimed to assess whether, in addition to SAP, HT also has an important mediating role in the association between eosinophilia and poor outcomes. With this study, we may obtain the following two new observations. First, the relationship between eosinophils and HT in patients with AIS undergoing mechanical thrombectomy (MT) was revealed. Second, the finding that HT may partially mediate the negative relationship between eosinophils and poor outcome. We expect that all these results will provide new ideas to unravel the mechanisms of cerebral ischemia-reperfusion injury and HT.
Consecutive patients with AIS who received endovascular treatment in our neurology department between December 2017 and March 2021 were prospectively recruited. Selection criteria for patients receiving first-line therapy, including direct aspiration, stent retriever, or a combination of stent retriever and local aspiration catheter, have been previously published [1]. To retrospectively analyze the relationship between eosinophils, HT, and functional outcomes, the exclusion criteria were. (1) patients receiving intra-arterial thrombolytic therapy only (n = 56); (2) asthma, eosinophilic esophagitis, hypereosinophilic syndrome, evidence of active infection, chronic inflammation, autoimmune disease, steroid therapy, cancer, hematologic disease, severe liver and kidney dysfunction (n = 4); and (3) unavailability of complete blood counts, medical records, or loss to follow-up (n = 9). A total of 328 patients with AIS met the study selection criteria and were included in the analysis (participant selection flowchart: Supplementary Fig. 1 in the supplementary file). The study protocol was approved by the Ethics Committee of the Second Hospital of Soochow University, and written informed consent was obtained from all patients or their relatives.
History including demographics (age, sex), potential stroke risk factors (atrial fibrillation, hypertension, diabetes, hyperlipidemia, smoking and alcohol consumption status), stroke etiology, stroke severity, Alberta Stroke Program Early CT Score (ASPECTS) at admission. Intravenous thrombolysis pretreatment (IVT) on admission, pre-morbid modified Rankin Scale (mRS) score, site of occlusion, status of collateral circulation, time to reperfusion at symptom onset or last seen, and blood index. Risk factors for stroke were defined according to previously published criteria [5]. Etiological subtypes and stroke severity were recorded as previously described [1, 6]. The status of collateral circulation prior to thrombectomy was assessed using the American Society of Interventional and Therapeutic Neuroradiology/Association of Interventional Radiology scale [7]. At the final angiogram, reperfusion status was graded according to the modified Thrombolysis in Cerebral Infarction (mTICI) score, with successful reperfusion defined as a score of 2b or 3 [1, 6]. Peripheral venous blood samples were obtained on admission to measure eosinophil levels. Patients were followed up by telephone or outpatient visits for 3 months. A modified Rankin Scale score of 3–6 at month 3 after admission [1, 6] was considered poor outcome.
On admission, all patients underwent CT scans. CT examinations were repeated immediately after mechanical thrombectomy and at 24 to 48 hours, and another CT scan was performed immediately in case of rapid neurological deterioration to assess the presence of HT. CT images were reviewed by a neuroradiologist with extensive experience in acute stroke, keeping medical records confidential. PH was defined as hemorrhage with mass effect according to previously published criteria [8, 9]. sICH was defined as any hemorrhage in the brain on CT scan with neurological deterioration [8, 9].
Characteristics of patients with and without HT (PH or sICH) were compared using the Chi-squared test, Fisher exact test, or Mann-Whitney U test. Multivariate analysis of variance regression models were used to assess the relationship between eosinophilia and HT. We first included age and female gender in the model (model 1). Subsequently, variables were included in model 2 if they were associated with HT (P < 0.10) or if they changed the estimated effect of eosinophils on HT by more than 10%.Supplementary Table 2–5 in the supplementary file show the relationship between each confounder and the outcome of interest [1, 10, 11]. Generalized additive models (GAM) and dichotomous linear regression models were used to identify nonlinear relationships and to calculate the threshold effect of eosinophilia on HT. Likelihood ratio tests were used to assess the modifying and interactive effects of oxygen saturation and subgroup variables on HT. Finally, after controlling for potential confounders, mediation analysis was used to assess whether HT mediated the relationship between eosinophilia and functional outcome. In addition, we calculated the direct, indirect and overall effects of predictors on functional outcome through mediating variables (SAP/hemorrhagic transformation) [1, 12]. All statistical analyses were performed using EmpowerStats (http://www.empowerstats.com, X&Y Solutions, Inc., Boston, MA). Bilateral P values < 0.05 were considered statistically significant [1, 10, 11].
Most baseline characteristics were balanced between patients included and excluded in this study, except for excluded patients who had less AF, lower NIHSS, and a different stroke etiology (Supplementary Table 1 in the supplementary file). A total of 328 patients with AIS (median age: 68 years) who underwent mechanical thrombectomy were included in this study. The main baseline characteristics of the patients according to the presence or absence of PH or sICH are listed in Table 1. eosinophil levels were significantly lower in the PH/sICH group than in the no PH/sICH group (P < 0.001).
|
Characteristics |
No PH (n = 250) |
PH (n = 78) |
P |
No sICH (n = 270) |
sICH (n = 58) |
P |
|---|---|---|---|---|---|---|
|
Age, y; median (IQR) |
68.00 (56.00–76.00) |
67.00 (58.50–74.00) |
0.897 |
67.00 (56.00–75.00) |
70.00 (62.25-75.00) |
0.082 |
|
Female, n (%) |
100 (40.00%) |
43 (55.13%) |
0.019 |
114 (42.22%) |
29 (50.00%) |
0.278 |
|
Atrial fibrillation, n (%) |
100 (40.00%) |
47 (60.26%) |
0.002 |
114 (42.22%) |
33 (56.90%) |
0.041 |
|
Hypertension, n (%) |
170 (68.00%) |
55 (70.51%) |
0.676 |
180 (66.67%) |
45 (77.59%) |
0.104 |
|
Diabetes, n (%) |
45 (18.00%) |
16 (20.51%) |
0.619 |
50 (18.52%) |
11 (18.97%) |
0.937 |
|
Hyperlipidemia, n (%) |
88 (35.20%) |
27 (34.62%) |
0.925 |
96 (35.56%) |
19 (32.76%) |
0.685 |
|
History of stroke, n (%) |
36 (14.40%) |
17 (21.79%) |
0.121 |
40 (14.81%) |
13 (22.41%) |
0.154 |
|
Smoking, n (%) |
77 (30.80%) |
23 (29.49%) |
0.826 |
85 (31.48%) |
15 (25.86%) |
0.399 |
|
Drinking, n (%) |
58 (23.20%) |
15 (19.23%) |
0.462 |
62 (22.96%) |
11 (18.97%) |
0.507 |
|
Baseline NIHSS, median (IQR) |
15.00 (12.00–18.00) |
18.50 (16.00–23.00) |
< 0.001 |
15.00 (12.00–19.00) |
19.00 (16.00-23.75) |
< 0.001 |
|
ASPECTS, median (IQR) |
7.00 (7.00–8.00) |
7.00 (6.00–7.00) |
0.009 |
7.00 (7.00–8.00) |
7.00 (6.00–7.00) |
0.003 |
|
Occluded artery, n (%) |
0.074 |
0.002 |
||||
|
ICA |
46 (18.40%) |
24 (30.77%) |
47 (17.41%) |
23 (39.66%) |
||
|
M1 of the MCA |
154 (61.60%) |
45 (57.69%) |
172 (63.70%) |
27 (46.55%) |
||
|
Posterior circulation |
32 (12.80%) |
5 (6.41%) |
31 (11.48%) |
6 (10.34%) |
||
|
Others |
18 (7.20%) |
4 (5.13%) |
20 (7.41%) |
2 (3.45%) |
||
|
IVT, n (%) |
78 (31.20%) |
25 (32.05%) |
0.888 |
85 (31.48%) |
18 (31.03%) |
0.947 |
|
Premorbid mRS, median (IQR) |
0.00 (0.00–0.00) |
0.00 (0.00–0.00) |
0.321 |
0.00 (0.00–0.00) |
0.00 (0.00–0.00) |
0.425 |
|
Stroke etiology, n (%) |
0.028 |
0.106 |
||||
|
LAA |
119 (47.60%) |
24 (30.77%) |
124 (45.93%) |
19 (32.76%) |
||
|
Cardioembolic |
118 (47.20%) |
50 (64.10%) |
131 (48.52%) |
37 (63.79%) |
||
|
Others |
13 (5.20%) |
4 (5.13%) |
15 (5.56%) |
2 (3.45%) |
||
|
Collateral score, median (IQR) |
0.00 (0.00–2.00) |
0.00 (0.00–1.00) |
0.183 |
0.00 (0.00–2.00) |
0.00 (0.00–1.00) |
0.015 |
|
OTR, median (IQR), min |
343.00 (277.75–440.00) |
337.00 (271.00-414.00) |
0.475 |
344.50 (278.75-440.25) |
340.50 (267.00-407.50) |
0.316 |
|
Number of passes, median (IQR) |
2.00 (1.00–3.00) |
2.00 (1.00–3.00) |
0.015 |
2.00 (1.00–3.00) |
2.00 (1.00–3.00) |
0.068 |
|
mTICI score 2b or 3, n (%) |
217 (86.80%) |
72 (92.31%) |
0.190 |
238 (88.15%) |
51 (87.93%) |
0.963 |
|
Eosinophils, 109/l; median (IQR) |
0.01 (0.00-0.04) |
0.00 (0.00-0.02) |
< 0.001 |
0.01 (0.00-0.04) |
0.00 (0.00–0.00) |
< 0.001 |
|
mRS score |
3.00 (1.00–4.00) |
6.00 (4.00–6.00) |
< 0.001 |
3.00 (1.00–4.00) |
6.00 (5.00–6.00) |
< 0.001 |
|
Poor outcome |
141 (56.40%) |
70 (89.74%) |
< 0.001 |
156 (57.78%) |
55 (94.83%) |
< 0.001 |
| ASPECTS, Alberta Stroke Program Early CT Score; IQR, interquartile range; ICA, internal carotid artery; IVT, intravenous thrombolysis; MCA, middle cerebral artery; mRS, modified Rankin Scale; mTICI, modified Thrombolysis in Cerebral Infarction; NIHSS, National Institutes of Health Stroke Scale; OTR, onset to reperfusion time; Posterior circulation, including basilar artery and intracranial part of the vertebral artery. | ||||||
Univariate analysis showed that AF, baseline NIHSS, ASPECTS, occluded artery, premorbid mRS, and collateral scores were positively associated with sICH, whereas ASPECTS, other stroke etiologies, and collateral scores were negatively associated with sICH (Supplementary Table 3 in the supplementary file). Univariate analysis results for PH were similar to those for sICH (Supplementary Table 4 in the supplementary file).
Table 2 summarizes the results of multivariate regression analysis. Eosinophils were independently associated with sICH as a continuous variable with an adjusted odds ratio (OR) of 0.00 (95% confidence interval (CI), 0.00–0.00; P = 0.0012) after adjusting for age and female (model 1) and 0.00 (95% CI, 0.00-0.01; P = 0.0141) after adjusting for all potential covariates (model 2). For sensitivity analysis, we transformed eosinophils into categorical variables by dichotomization, and the OR (95% CI) for sICH was 0.22 (0.11–0.46) for participants with higher eosinophils compared with patients with lower percentages of eosinophils. For the test of binary logistic regression of PH, a similar association was found between eosinophils and PH. Higher eosinophilia (OR 0.36, 95% CI: 0.19–0.67; P = 0.0013) remained an independent predictor of decreased risk of PH after adjusting for all potential covariates (model 2).
|
PH OR; (95% CI); P Value |
sICH OR (95% CI) |
|||||
|---|---|---|---|---|---|---|
|
Non-adjusted model |
Model 1 |
Model 2 |
Non-adjusted model |
Model 1 |
Model 2 |
|
|
Eosinophils |
0.00; (0.00, 0.00); 0.0018 |
0.00; (0.00, 0.00); 0.0027 |
0.00; (0.00, 0.05); 0.0174 |
0.00; (0.00, 0.00); 0.0008 |
0.00; (0.00, 0.00); 0.0012 |
0.00; (0.00, 0.01); 0.0141 |
|
Eosinophils (dichotomous) |
0.32; (0.19, 0.54); <0.0001 |
0.33; (0.19, 0.56); <0.0001 |
0.36; (0.19, 0.67); 0.0013 |
0.18; (0.09, 0.34); <0.0001 |
0.18; (0.10, 0.35); <0.0001 |
0.22; (0.11, 0.46); <0.0001 |
| Non-adjusted model: we did not adjust other covariates. | ||||||
| Model 1: we adjusted age and female. | ||||||
| Model 2: we adjusted variables which were significantly associated with outcomes of interest (P < 0.10) or changed the estimates of eosinophils on outcomes of interest by more than 10% (Supplementary Table 2–7 in the supplementary file). | ||||||
| CI indicates confidence interval; OR, odds ratio; PH, parenchymal hematoma; sICH, symptomatic intracranial hemorrhage. | ||||||
In the present study, we analyzed the nonlinear relationship between eosinophils and sICH/PH (Supplementary Fig. 2 in the supplementary file). No nonlinear relationship was found between eosinophils and sICH/PH (Supplementary Fig. 2–3 in the supplementary file). In addition, there were no statistically significant inflection points in the threshold effects analysis (P of 1 for the log-likelihood ratio test, suggesting that standard linear regression rather than dichotomous linear regression may be more appropriate for analyzing the relationship between eosinophils and sICH). Subgroup analysis further confirmed these associations between eosinophils and sICH. As shown in Supplementary Table 10 in the supplementary file, the interaction tests for age, AF, diabetes mellitus, and hyperlipidemia were statistically significant (P values for the interactions were less than 0.05). Similar associations were found between eosinophilia and PH (Supplementary Table 10 in the supplementary file).
Eosinophils were also associated with poor outcome (as a continuous variable. OR, 0.00; 95% CI, 0.00-0.09; P = 0.0086; as a categorical variable. OR, 0.21; 95% CI, 0.10–0.46; P < 0.0001. Table 2 and Supplementary Table 8 in the supplementary file), which is consistent with previous studies [1, 13–15]. We then constructed a hypothetical model of the relationship between eosinophils, HT, and functional outcome. Our results suggest that sICH partially mediates the relationship between eosinophils and poor outcome (Fig. 1). The proportion of the total effect of eosinophils on poor outcomes mediated by sICH was 10.36% (95% CI, 1.82%-27%). After removing the effects mediated by sICH, the direct effect of eosinophils on poor outcomes (total effect minus indirect effect) remained statistically significant (P < 0.001). However, no mediating effect of PH between eosinophilia and poor outcomes was found in this study (Supplementary Fig. 4 in the supplementary file).
Endovascular therapy is clinically effective and offers cost savings for patients with AIS compared with usual care alone. HT is a serious complication after endovascular therapy for ischemic stroke, which increases the risk of death at 3 months and the likelihood of poor functional outcome [8, 16, 17]. The present study was performed to assess the relationship between eosinophils, HT and functional outcome after mechanical thrombectomy in patients with AIS. And the present study showed that decreased eosinophil levels were associated with an increased risk of sICH/PH. In addition, there was an association between eosinophils and functional outcome, which is consistent with previous studies [13–15]. More importantly, this study showed an important role of sICH on this association.
Many risk factors such as neutrophils and neutrophil-to-lymphocyte ratio have been identified for sICH [8, 18, 19]. To our knowledge, no study has investigated the effect of eosinophils on sICH after mechanical thrombectomy. In the present study, a decrease in eosinophils was associated with an increase in sICH (OR, 0.00; 95% CI, 0.00-0.01; P = 0.0141). The mechanisms underlying these observations are not well established, but eosinophil-induced neuroprotection may play a critical regulatory role in the development of sICH. Eosinophils can secrete vascular endothelial growth factor and several chemokines [2–4]. IL-4 and IL-13 secreted by eosinophils can induce the activation of M2 phenotype microglia, which are neuroprotective by promoting the resolution of inflammation. In contrast, vascular endothelial growth factor may have neuroprotective effects by regulating angiogenesis [2–4]. Alternatively, a decrease in eosinophils may lead to reduced neuroprotection and more sICH, which in turn leads to a worse outcome (Supplementary Fig. 7 in the supplementary file).
Eosinophils are associated with both sICH and functional outcome. sICH partially mediates the relationship between decreased eosinophil levels and poor outcome. These data suggest that eosinophil-induced neuroprotection and subsequent complications of sICH may be one of the mechanisms underlying the association between eosinophils and functional outcome. Furthermore, we found a significant association of eosinophils with poor outcome independent of sICH in multivariate analysis (mRS: β, -5.00; 95% CI, -8.71-1.30; P = 0.0086; poor outcome: OR, 0.00; 95% CI, 0.00-0.09; P = 0.0086. Supplementary Table 11 in the supplementary file). These results suggest that eosinophils have additional prognostic value when considering sICH, and in addition to sICH, eosinophils may contribute to adverse outcomes such as stroke-associated pneumonia (Supplementary Fig. 6 in the supplementary file).
A previous study showed that ischemic stroke decreased the proportion of eosinophils in the lungs [20]. The altered eosinophils coincided with a marked reduction in the levels of multiple chemokines and cytokines in the lungs that are essential to "pre-condition" the lungs against bacterial infection or to promote bacterial clearance in the event of lung infection [1, 17]. These suggest that ischemic stroke creates an immunosuppressive environment in the lungs by reducing the production of multiple pro-inflammatory chemokines and cytokines. In addition, extracellular structures formed by mitochondrial DNA and granule proteins released by eosinophils are able to bind and kill bacteria, which contributes to antimicrobial defense [1, 20, 21]. Whereas our previous study found that lower eosinophil levels were associated with higher SAP, SAP plays an important mediating role in the association of eosinophils with adverse outcomes. Given the above, it is possible that a decrease in eosinophils could lead to more sICH and SAP, which in turn could lead to poor outcomes (Supplementary Fig. 7 in the supplementary file). To test our hypothesis, further animal studies are needed.
Although eosinophilia was also associated with PH (OR, 0.00; 95% CI, 0.00-0.05; P = 0.0174), no mediation effect of PH on the association between eosinophilia and poor outcome was found in the present study. The reason for this discrepancy may be related to the small sample size, which weakens the statistical strength of our conclusions. Another reason for the mediating effect of sICH rather than PH may be that poor outcome itself is a clinical definition. sICH reflects the degree of brain injury and clinical symptoms, whereas PH reflects only the degree of brain injury, which leads to a situation where sICH better reflects functional outcome. And we did find that eosinophils appeared to be more sensitive to sICH (OR, 0.00; 95% CI, 0.00-0.01; P = 0.0141) or sICH to poor outcome (OR, 9.06; 95% CI, 2.31–35.48; P = 0. 0015) than eosinophils to PH (OR, 0.00; 95% CI, 0.00-0.05; P = 0.0174) or the effect size of PH on poor outcomes (OR, 6.27; 95% CI, 2.39–16.46; P = 0.0002). And further studies from other larger samples of patients with AIS are needed to explore whether PH mediates the association between eosinophilia and poor outcomes.
The main strength of our study is that we provide a comprehensive study with the established model to assess the effect of sICH on the relationship between eosinophil and functional outcomes. However, this study also has some limitations. First, eosinophil levels were measured at only one point, and therefore, results may vary depending on the possible rapid changes in their values after the onset of symptoms [8, 22]. Second, we neither explored the mechanisms by which eosinophils affect immunosuppressive and neuroprotective pathways nor investigated which factors regulate changes in eosinophils after ischemic stroke in animal studies. These will be the focus of our next work, especially to explore the role of eosinophils in ischemic stroke and their mechanisms. Third, this is a retrospective single-center study with a small sample size. In addition, our cohort represents a subgroup of stroke patients who underwent thrombectomy; therefore, our results may not be generalizable to the entire stroke patient population and further studies from other samples of AIS patients are needed to validate our results. Fourth, our study was designed as a cross-sectional study; therefore, causality could not be determined. To compensate for this limitation, we performed a causal mediation analysis and suggested a possible association between eosinophilia, sICH, and functional outcome (Fig. 1; Supplementary Figs. 4–6 in the supplementary file). However, it is noteworthy that this is the first study to show a complex relationship between eosinophils, sICH, and functional outcome in patients with AIS experiencing MT. In this regard, attempts to maintain eosinophils have important implications for stroke prognosis, especially considering that clinical outcomes at 3 months remain unsatisfactory, with nearly half of patients successfully reperfused presenting with an unfavorable functional prognosis [23].
The present study shows that reduced eosinophil levels are associated with a high risk of sICH and poor outcome in patients with AIS experiencing MT, and that sICH is on the pathway of association between eosinophils and functional outcomes. However, PH did not mediate the association between eosinophils and poor outcome.
|
AIS |
Acute ischemic stroke |
|
AF |
atrial fibrillation |
|
ASPECTS |
Alberta Stroke Program Early CT Score |
|
CI |
Confidence interval |
|
HT |
Hemorrhagic transformation |
|
IQR |
Interquartile range |
|
IVT |
Intravenous thrombolysis |
|
mRS |
modified Rankin Scale |
|
MT |
mechanical thrombectomy |
|
mTICI |
modified Thrombolysis in Cerebral Infarction |
|
NIHSS |
National Institutes of Health Stroke Scale |
|
OR |
Odds ratio |
|
PH |
parenchymal hematoma |
|
SAP |
stroke-associated pneumonia |
|
sICH |
symptomatic intracranial hemorrhage |
Acknowledgments
Not applicable.
Authors Contributions
SY and XW contributed to the concept and rationale for the study. SY and YL were responsible for the first draft. ZLG contributed statistical analyses. HH and ZLG performed the data collection and curation. ZLG and YL contributed to the first revision. All authors read and approved the final manuscript.
Funding Sources
Suzhou Medical and Health Science and Technology Innovation (SKY2022160).
Data Availability
The raw data supporting the conclusions of this article will be made available by the authors (Zhiliang Guo), without undue reservation.
Ethics approval and consent to participate
The study was carried out according to the Declaration of Helsinki and the Guideline for Good Clinical Practice. The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Soochow University, and written informed consent was obtained from all patients or their relatives.
Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Conflict of Interest
The authors declare that there is no confict of interests.