Correlation of common inflammatory cytokines with cognition impairment, anxiety, and depression in acute ischemic stroke patients

Abstract Inflammatory cytokines are related to cognitive function and psychiatric disorders in patients with several diseases. However, few relevant studies have been performed on acute ischemic stroke (AIS) patients. Hence, this study aimed to investigate the correlation of common inflammatory cytokines with cognition impairment, anxiety, and depression in AIS patients. Common inflammatory cytokines of 176 AIS patients (including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-17) were measured using Human Enzyme Linked Immunosorbent Assay Kits. Cognition impairment (Mini-Mental State Examination (MMSE)), anxiety (Hospital Anxiety and Depression Scale for anxiety (HADS-A)), and depression (HADS-D) were evaluated. The incidence of cognition impairment, anxiety, and depression was 43.2, 39.2, and 31.2%, respectively. TNF-α and IL-6 were negatively associated with MMSE score, and high TNF-α, IL-1β, and IL-6 were correlated with cognition impairment occurrence. In addition, TNF-α, IL-1β, and IL-17 were positively associated with HADS-A score, while only high TNF-α was associated with anxiety occurrence. Furthermore, TNF-α, IL-1β, and IL-17 were positively associated with HADS-D score, while high IL-1β, IL-6, and IL-17 correlated with depression occurrence. Multivariate logistic regression revealed that TNF-α and National Institutes of Health Stroke Scale (NIHSS) score ≥5 were associated with high risk of cognition impairment; TNF-α, IL-17, unemployed before surgery, hypertension, and chronic kidney disease (CKD) correlated with high anxiety occurrence. Furthermore, IL-17, divorced/widowed/single status, diabetes, and NIHSS score ≥5 were associated with high risk of depression. In conclusion, common inflammatory cytokines including TNF-α, IL-1β, and IL-17 were related to cognition impairment, anxiety, or depression in AIS patients.


Introduction
Stroke is a huge health challenge characterized by the dysfunction and degeneration of brain vascular components, with over 2.5 million new cases in China annually (1). Acute ischemic stroke (AIS) is a major pathological type of stroke and occupies nearly 80% of new stroke cases (2,3). Although the recent decades have witnessed substantial advances in diagnostic and treatment options, AIS is still the leading cause of disability and one of the leading causes of mortality worldwide (4). Of note, several severe complications often occur to AIS patients, among which, cognitive impairment is a sort of neurological dysfunction manifestation presenting with dysmnesia, disorientation, decline of language competence, and so on, which directly decreases the quality of life of AIS patients (5,6). Other common psychological complications of AIS patients such as anxiety and depression have a negative influence on rehabilitation outcomes, neurotrophic agents' efficacy, and even mortality in AIS patients (7). Therefore, investigating potential indicators related to cognition impairment, anxiety, and depression is a prerequisite for developing effective therapies to improve outcomes of AIS patients.
Inflammation is considered a key step of the progression of ischemic stroke, which has been shown to increase brain injury, retard brain repair, and affect neurological outcomes (8,9). Interestingly, inflammation is also related to cognition impairment, anxiety, and depression in patients with several diseases. For instance, high serum creatinine-reactive protein is associated with depression occurrence in patients with isolated coronary artery ectasia (10). Also, increased TNF-a and IL-6 are correlated with high risk of mild cognitive impairment in type 2 diabetes patients (11). However, few relevant studies have been performed in AIS patients. Hence, this study aimed to investigate the correlation of common inflammatory cytokines with cognition impairment, anxiety, and depression in AIS patients.

Patients
This study obtained approval from the Institutional Review Board of The Second Affiliated Hospital of Harbin Medical University. Between January 2019 and July 2020, 176 AIS patients who were admitted to this hospital were consecutively recruited for this study. Eligibility criteria for recruitment were as follows: i) diagnosis of AIS in line with the AIS guideline (12); ii) age more than 18 years; iii) volunteer to participate in the study and provide a blood sample for study use; and iv) able to complete the study assessment. Patients with any of the following conditions were considered ineligible for study enrollment: i) severe cognitive impairment, which was defined as Mini-Mental State Examination (MMSE) score o10; ii) presenting with intracranial hemorrhage; iii) known hematological diseases or active infections; iv) administered immunosuppressant within 3 months; v) complicated with inflammatory diseases; vi) had a history of malignancies; and vii) breast feeding or pregnant. All patients signed an informed consent prior to recruitment.

Sample collection and analysis
Venous blood samples of patients were collected before they were discharged from the hospital after 12-h fasting, and the serum was separated within two hours. After blood collection, the tube was gently inverted and mixed 4-5 times, then put in the upright position at room temperature until the blood was completely coagulated (about 1 h). Following that, centrifugation was conducted at 1000 g for 10 min at room temperature, then serum was obtained. The collected serum was transferred to cryopreserved tubes and placed in a -70°C refrigerator. Levels of inflammatory cytokines in serum, including TNF-a, interleukin (IL)-1b, IL-6, and IL-17, were measured using Human Enzyme Linked Immunosorbent Assay (ELISA) kits (Invitrogen, USA). The ELISA was carried out following the manufacturer's protocol.

Data collection and evaluation
Clinical data collection was completed during hospitalization of patients, which mainly included sociodemographic information, smoking status, complications, as well as disease-related features. Assessment of cognition impairment, anxiety, and depression was conducted for all patients on the day of discharge. The cognition impairment status of patients was evaluated using MMSE, and a MMSE score p26 was considered as cognition impairment (13). The anxiety status and depression status of patients were assessed using the Hospital Anxiety and Depression Scale for anxiety (HADS-A) and the Hospital Anxiety and Depression Scale for depression (HADS-D), respectively. An HADS-A score 47 was indicative of anxiety, and similarly, a HADS-D score 47 was indicative of depression. Furthermore, the anxiety degree was classified as 8-10, mild anxiety; 11-14, moderate anxiety; and 15-21, severe anxiety (14). The depression degree was classified as 8-10, mild depression; 11-14, moderate depression; and 15-21, severe depression (14).

Statistical analysis
SPSS 24.0 (IBM, USA) and GraphPad Prism 7.02 software (GraphPad Software Inc., USA) were applied for data analysis and diagram making. Descriptive analysis was performed for characteristics of patients, MMSE score, HADS-A score, and HADS-D score. Correlation analysis was determined by Spearman's rank correlation test. Comparison of differences was determined by chisquared test or Wilcoxon rank sum test, as appropriate. All potential factors were included in the multivariate logistic regression analysis of cognition impairment, anxiety, and depression, and the independent factors were screened out by forward stepwise (conditional) method. A significant difference was indicated by a P value o0.05.

AIS patients' characteristics
The detailed information about other characteristics is shown in Table 1. The mean age was 67.6 ± 8.4 years, and there were 63.1% males and 36.9% females. With respect to marriage status, 47.2% patients were married and 52.8% patients were divorced/widowed/single. As for employment status before AIS, 11.4% patients were employed and 88.6% patients were unemployed. In addition, 27.3% patients were current smokers, 84.7% patients had hypertension, 50.0% patients had hyperlipidemia, 36.4% patients had diabetes mellitus, and 13.6% patients had CKD. Furthermore, 40.3, 34.1, and 25.6% of patients had left, right, and bilateral brainstem unknown lesion location, respectively. The mean NIHSS score was 7.0±3.0.

Discussion
In the current study, the incidence of cognition impairment, anxiety, and depression was 43.2, 39.2, and 31.2% in AIS patients. After acute ischemic insult, inflammatory cytokines in the ischemic brain will be upregulated from resident brain cells and infiltrating immune cells, which play complex roles in the pathophysiology of cerebral ischemia (9). Previous evidence supports a relationship of cognition impairment, anxiety, and depression with inflammation processes in several diseases (such as coronary heart patients and cancer) (10,15,16). However, few studies have been performed on the potential cognition impairment/anxiety/depression-associated inflammatory cytokines in AIS patients. Hence, deeply understanding the clinical implication of inflammatory cytokines on cognition impairment/anxiety/depression is a precondition to improve the prognosis of AIS patients. In the present study, we discovered that high TNF-a and IL-6 were correlated with cognition impairment occurrence; high TNF-a, IL-1b, and IL-17 were correlated with anxiety or depression occurrence in AIS patients. The possible reasons were that: 1) TNF-a and IL-6, as pro-inflammatory cytokines, could cross the blood-brain barrier by a transport system, thereby promoting communication between the central nervous system and the periphery (17,18). Hence, high concentrations of TNF-a and IL-6 might be important factors for promoting the development of cognition impairment, thereby increased cognition impairment occurrence; and 2) a high level of common pro-inflammatory cytokines (including TNF-a, IL-1b, and IL-17) might affect indoleamine 2,3-deoxygenation enzyme-1 (IDO1), subsequently influence 5-hydroxytryptamine (5-HT) (an important neurotransmitter related to depression), and eventually increase anxiety and depression occurrence in AIS patients (19)(20)(21)(22).
In addition, TNF-a and NIHSS score X5 were related to a high risk of cognition impairment. Also, IL-17, unemployed before surgery, hypertension, CKD, divorced/ widowed/single status, diabetes, and NIHSS score X5 were correlated with a high risk of anxiety or depression in AIS patients. The probable explanations were as follows: 1) Regarding cognition impairment, TNF-a served as a common inflammation cytokine, and the possible reason for its correlation with cognition impairment was as mentioned above; high NIHSS score meant worse neurological status in AIS patients, and cognition impairment was also a kind of manifestation of neurological dysfunction, hence, an NIHSS score X5 was an independent risk factor for cognition impairment. 2) As for anxiety and depression, the impact of IL-17 on anxiety and depression was also as mentioned above. Furthermore, unemployed before surgery and divorced/widowed/single status meant unfavorable social status, which might make patients feel less capable  and alone, increasing stress after AIS, thereby causing a high risk of anxiety and depression. Meanwhile, patients with severe complications (including hypertension, CKD, and diabetes) are under long-term metabolic dysfunction, which might affect their immune system and increase their negative stress, thereby leading to a high risk of anxiety and depression in AIS patients. In addition, an NIHSS score X5 meant worse neurological status in AIS patients, which also produces huge stress, and eventually, increased anxiety and depression occurrence in AIS patients.    SE: standard error; OR: odds ratio; CI: confidence interval; TNF-a: tumor necrosis factor alpha; NIHSS: National Institutes of Health Stroke Scale; IL-17: interleukin 17; CKD: chronic kidney disease. *NIHSS score was evaluated within 24 h after admission. # All potential factors were included in the multivariate logistic regression analysis, and the independent factors were screened out by forward stepwise (conditional) method.
Several limitations existed in this study. Although the potential cognition impairment/anxiety/depression-associated inflammatory cytokines in AIS patients have been explored, the detailed mechanisms of these inflammatory cytokines underlying cognition impairment/anxiety/ depression of AIS patients still remain unclear. Further relevant study is needed.
In conclusion, common inflammatory cytokines including TNF-a, IL-1b, and IL-17 were related to cognition impairment, anxiety, and depression in AIS patients in this study.