Neutrophil‐to‐lymphocyte ratio, hyperglycemia, and outcomes in ischemic stroke patients treated with intravenous thrombolysis

Abstract Introduction Increased neutrophil‐to‐lymphocyte ratio (NLR) and hyperglycemia on admission are associated with poor outcomes in acute ischemic stroke (AIS) patients. We sought to evaluate the combined effect of increased NLR and hyperglycemia on the prognosis of ischemia stroke treated with intravenous thrombolysis (IVT). Methods Patients with acute ischemic stroke receiving IVT treatment were prospectively enrolled. All participants were followed for 3 months. According to the levels of NLR and blood glucose, patients were categorized into four groups: high NLR or nonhigh NLR with or without hyperglycemia. The associations between NLR values with or without hyperglycemia and outcomes of stroke after thrombolysis were assessed by multivariable logistic regression analysis. Results Among the 381 stroke patients (median age 68 years, 61.68% man) included, 155 (40.68%) had a poor outcome (modified Rankin Scale score 3–6) during 3 months. After multivariate adjustment, high NLR with hyperglycemia increased the risk of 3‐month poor outcome (OR = 4.42; 95% CI, 2.13–9.16), early neurological deterioration (END) (OR = 4.81; 95% CI, 2.08–11.12), and 3‐month mortality (OR = 6.56; 95% CI, 1.92–22.40). A significant multiplicative interaction of NLR and blood glucose on 3‐month poor outcome in ischemic stroke patients after thrombolysis was observed. Conclusions Ischemic stroke patients with concurrent high NLR and hyperglycemia increased risks of END, 3‐month poor outcome, and mortality after thrombolysis.


| BACKG ROU N D
Acute ischemic stroke (AIS) is a grave threat to global healthcare system, which is accompanied by high disability and mortality (Fan, Gui, Chai, & Wei, 2018). Intravenous thrombolysis with recombinant tissue plasminogen activator (rtPA) is considered as the most effective medical reperfusion treatment within 4.5 hr after the onset of symptoms in patients with AIS (Emberson et al., 2014). However, increased risks of side effects following IVT such as cerebral hemorrhage (HT) may partially offset the benefits of reperfusion and even lead to neurological deterioration (Jickling et al., 2014;Seners et al., 2014).
Therefore, the detection of useful biomarkers is essential to an early risk assessment and effective therapy after thrombolysis.
Recently, mounting attention has been drawn to the prognostic role of inflammation in AIS. AIS is no longer believed to be only a vascular disease, but also an immune-mediated condition (Jiang, Moyle, Soule, Rote, & Chopp, 1995). Neutrophil, a critical immune responder to ischemic brain insult, has been suggested to accumulate accompanying progression of infarct lesion and exacerbate brain damage (Ishikawa, Zhang, Nanda, & Granger, 2004;Jiang, Moyle, Soule, Rote, & Chopp, 1995). In addition, neutrophil was associated with the higher disability and mortality rates in stroke patients receiving rtPA treatment Malhotra et al., 2018). Similarly, neutrophil-to-lymphocyte ratio (NLR) has been widely investigated and has emerged as a novel inflammation marker. Previous studies have demonstrated NLR as a predictor of major disability , mortality (Tokgoz, Keskin, Kayrak, Seyithanoglu, & Ogmegul, 2014), and HT (Guo et al., 2016). Admission hyperglycemia is prevalent in more than a third of AIS patients (Dungan, Braithwaite, & Preiser, 2009) and plays an important role in prognosis of stroke. Increased risks of poor functional outcomes, mortality, and HT have been observed for high admission blood glucose level in stroke patients treated with IVT (Lin et al., 2018;Poppe et al., 2009). Moreover, several studies have suggested the deleterious effects of hyperglycemia are attributable to the reperfusion following thrombolysis (Alvarez-Sabín et al., 2004;Putaala et al., 2011). Interestingly, hyperglycemia has been shown to enhance endothelial activation and neutrophil infiltration in stroked rats (Justicia et al., 2017). A clinical study has demonstrated a strong association between NLR and hyperglycemia in patients with spontaneous intracerebral hemorrhage . However, the combined relationship between NLR and hyperglycemia in AIS patients after rtPA treatment has not been well studied.
In the present study, we conducted a prospective study to investigate the combined effects of the pattern of high NLR and hyperglycemia on poor outcomes in ischemic stroke patients after thrombolysis.

| Study population
Consecutive AIS patients at the First Affiliated Hospital of Wenzhou Medical University from January 2016 to January 2019 were prospectively enrolled in our study. The inclusion criteria for enrollment were (a) older than 18 years, (b) diagnosis of AIS and treatment with IV rtPA within 4.5 hr of symptom onset. Exclusion criteria included (a) evidence of acute infection at admission or any infection that occurred during the first 48h after admission; (b) cancer, chronic inflammation disease, autoimmune disease, or immunosuppressive drug use; and (c) patients who received a bridging therapy consisting of IV rtPA followed by endovascular therapy. Finally, a total of 381 ischemic stroke patients were included in the current study. The present study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University, and all patients or their relatives gave informed consent.

| Baseline assessment
Baseline information for all patients including demographics, risk factors (blood pressure, smoking, and alcohol drinking status), onsetto-treatment time, and medical history (diabetes, hypertension, dyslipidemia, atrial fibrillation, and history of stroke) were recorded on admission. Stroke severity was assessed by trained neurologists with the National Institutes of Health Stroke Scale (NIHSS) on admission.
Venous blood samples were collected within 24 hr of hospital admission after overnight fasting. A complete blood count was determined using the automated hematology analyzer (XE-2100, Sysmex Company, Japan) within 60 min after blood sample collection. NLR was calculated as the neutrophil count divided by the lymphocyte count.
Hyperglycemia was defined as a fasting glucose level of ≥110 mg/dl (6.1 mmol/L). Diabetes mellitus was defined as HbA1c ≥ 6.5% or current use of oral or parenteral antidiabetic medicines. Hypertension was defined as a systolic blood pressure ≥140 mm Hg or a diastolic pressure ≥90 mm Hg persisting after the acute stage of ischemic stroke, or by antihypertensive medication use prior to admission. Dyslipidemia was defined as a fasting plasma cholesterol level ≥220 mg/dl, a fasting plasma triglyceride level ≥150 mg/dl, or by the use of lipid-lowering medications prior to admission.

| Outcome measures
The primary outcome was poor functional outcome (modified Rankin Scale (mRS) score, 3-6) at 3 months after stroke. Secondary outcomes were early neurological deterioration (END) and 3-month mortality. END was defined as an increment of at least 4 point within 24 hr following IVT compared to the NIHSS scores on admission (Seners et al., 2014). All patients were followed up prospectively by telephone interview at 3 months conducted by a trained neurologist.

| Statistical analysis
The results were presented as the mean (standard deviation, SD) or median (interquartile range, IQR) for continuous variables and percentages for categorical variables. NLR at baseline was categorized into two levels according to NLR median value in all participants (High NLR ≥ 4.0; Nonhigh NLR < 4.0). The study patients were then classified into four groups in accordance with their NLR and fasting glucose levels: nonhigh NLR and nonhyperglycemia (Non-HR and non-HG), nonhigh NLR and hyperglycemia (Non-HR and HG), high NLR and nonhyperglycemia (HR and non-HG), and high NLR and hyperglycemia (HR and HG). The differences among four groups were tested by one-way analysis of variance (ANOVA) or the Kruskal-Wallis H test for continuous variables, as appropriate.
Comparisons of two groups in terms of continuous variables were evaluated with Student's t test and Mann-Whitney U test as appropriate. Categorical variables were compared using the chi-square test. Logistic regression analysis was performed to estimate the probability of having poor outcomes of stroke after IVT and 95% CI for each risk factor category stratified by NLR and glucose levels, adjusting for age, gender, and other conventional confounders. We tested the statistical significance of NLR category × glucose status in multivariable logistic model to examine the multiplicative interaction of elevated NLR and hyperglycemia.

| RE SULTS
In this study, a total of 533 AIS patients treated with rtPA were screened, and 152 patients were excluded due to occurrence of acute infection (n = 68), bridging therapy (n = 27), a history of cancer (n = 8), autoimmune disease or immunosuppressive drug use (n = 14), and losing to follow up (n = 35).
Of the 381 patients in the study sample (median age 68 years, 61.68% man), 155 (40.7%) had poor outcome at 3 months after IVT.  Figure 1). When patients were categorized into four groups according to the levels of NLR and blood glucose, the subjects with both high NLR and hyperglycemia accounted for 24.4% of the total studied population. These subjects were more likely to be elderly and have vascular risk factors including hypertension, hyperlipidemia, and diabetes (Table 2). In patients with high NLR and hyperglycemia, there is an increase in the scores of 4, 5, and 6, but a decrease in the scores of 0, 1, and 2 ( Figure 2).
The association of NLR and blood glucose with clinical outcomes after thrombolysis was presented in Table 3. In the univariate analysis, patients with both high NLR and hyperglycemia increased the risks of END, 3-month poor outcome, and mortality (all p < .05), compared to those with low NLR and blood glucose. This effect was consistent after adjusting for age, sex, and other potential confounding factors. However, low NLR with hyperglycemia and high NLR without hyperglycemia were not linked to any poor outcomes in this study. Furthermore, the interactive effect of the pattern of high NLR and presence of hyperglycemia on the 3-month poor outcome was observed (P for interaction = 0.035). High NLR was merely a strong predictor of 3-month poor outcome (adjusted OR (aOR) = 4.12; 95% CI, 1.57-10.75) and END (aOR = 4.30; 95% CI, 1.38-13.38; Figure 3) in stroke patients with hyperglycemia.

| D ISCUSS I ON
The study investigated the association of NLR and blood glucose on A meta analysis also has shown that admission glucose level >6.1 mmol/L has an increased risk of 30-day mortality in nondiabetic patients after stroke (Capes et al., 2001). On the other hand, neutrophils are generally the first and most abundant inflammatory cells to respond to ischemic stroke and migrate to cerebral ischemic regions during the whole acute phase of inflammation (Jin, Yang, & Li, 2010;Shichita, Sakaguchi, Suzuki, & Yoshimura, 2012). Prior studies found that neutrophil count was related to stroke severity at admission (Kim et al., 2012), SICH, and 3-month mortality (Maestrini et al., 2015) in patients with stroke. Neutrophil-to-lymphocyte ratio (NLR) emerged as a systemic biomarker for inflammation. In terms of ischemic stroke, NLR has been identified as an independent predictor of unfavorable clinical outcomes (Qun et al., 2017). Jijun Shi at al.
found that NLR was a dynamic variable and NLR at 24 hr after rtPA infusion was significantly associated with increased risks of death and major disability at 3 months (Shi et al., 2018). Experimental studies revealed that hyperglycemia enhanced neutrophil infiltration in the ischemic brain and exacerbated the brain lesion (Justicia et al., 2017;Lin, Ginsberg, Busto, & Li, 2000). In our study, elevated NLR was an independent risk indicator for poor outcome in stroke patients with hyperglycemia, while we did not observe any association between NLR and outcomes in patients without hyperglycemia.

F I G U R E 1
Comparison of outcomes between NLR values with or without hyperglycemia. HG, hyperglycemia; HR, high neutrophilto-lymphocyte ratio; mRS, modified Rankin Scale However, most of the previous studies did not offer such stratification. Our study found a significant interaction between NLR and blood glucose on 3-month poor outcome in ischemic stroke patients after IVT. It has to be mentioned that NLR was not further stratified based on the levels of glucose in this study, which might underestimate the association between elevated NLR and poor outcome in  Yu, Nito, & Chan, 2007). It is worth noting that neutrophils have been shown to be an important source of MMP-9 and may mediate HT through MMP-9 in ischemic stroke (Gautier et al., 2014).
As mentioned above, the pathological mechanisms of inflammatory responses and hyperglycemia on poor outcomes of AIS after IVT impact each other through several complex signal pathways, though the definite biological mechanisms are still insufficiently understood. Therefore, more large studies are needed to further investigate the precise mechanisms.

| Study limitations
Our study has several limitations. First, the study population was relatively small from a single hospital, which inevitably produced selection bias and may limit the generalization of our

| CON CLUS IONS
The combination of high NLR and hyperglycemia was a stronger predictor to 3-month poor functional outcome, 3-month mortality, and END in patients with acute ischemic stroke after IVT treatment.
Based on this finding, strict control of inflammatory parameters and glucose may help to improve outcome for patients with ischemic stroke.

ACK N OWLED G M ENTS
This study was supported by the National Natural Science Foundation of China (81673144, 81301014) and Foundation of Wenzhou Science and Technology Bureau (Y20160025).

CO N FLI C T O F I NTE R E S T
None declared.

AUTH O R CO NTR I B UTI O N S
Yani Cheng and Anna Ying conceptualized and designed the study, analyzed the data, and drafted the manuscript. Yanyan Lin and Junru Yu assisted in conceptualizing and designing the study. Ji Luo and Yifan Zeng collected data. YSL contributed to rewriting and editing the final version of the manuscript. All authors read and approved the manuscript.

PEER R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.1741.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

R E FE R E N C E S
F I G U R E 3 Adjusted ORs ratios of NLR levels for outcomes among ischemic stroke patients with or without hyperglycemia. Adjusted for age, sex, cigarette smoking, alcohol consumption, systolic blood pressure, onset-to-treatment time, hypertension, hyperlipidemia, atrial fibrillation, previous stroke, baseline NIHSS score, triglyceride, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol. CI, confidence interval; OR, odds ratio