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Serum Periostin and Cognitive Impairment in Ischemic Stroke Patients: a Prospective Cohort Study

Background. Recent evidence suggest elevated periostin is associated with cardiovascular diseases. The aim of this study was to investigate the relationship between serum periostin and post-stroke cognitive impairment (PSCI) at 3 months. Methods. In this prospective cohort study, we enrolled patients with ischemic stroke and hospitalized within 7 days of symptoms onset from January 2019 to January 2020. Serum periostin levels were measured using enzyme-linked immunosorbent assay after admission. Cognitive function assessment was performed at 3-month follow-up visit using the Montreal Cognitive Assessment (MoCA). We dened the PSCI as total MoCA score < 25. Results. A total of 315 ischemic stroke patients were enrolled for the study. PSCI was observed in 173 patients, which accounted for 54.9% (95% condence interval [CI] 52.1%–57.7%) of the cohort. Serum periostin levels were higher in patients with PSCI than in those without PSCI (median 19.6 vs 14.8 ng/mL; P = 0.001). In logistic regression analysis, the highest quartile of periostin levels were signicantly correlated to PSCI (odds ratio [OR], 9.69; 95% CI, 5.06–25.61; P = 0.001), as compared with the lowest quartile. This association remained signicant after adjustment for age, gender, educational years, stroke severity, and vascular risk factors. Subgroup analyses further conrmed these results. Furthermore, restricted cubic spline regression demonstrated a linear association between periostin levels and PSCI (P = 0.001 for linearity). Conclusions. This study found that higher serum periostin levels are associated with an increased risk of PSCI at 3 months after ischemic stroke onset.


Introduction
Post-stroke cognitive impairment (PSCI) is regarded as the most prevalent syndrome after stroke [1]. Reported frequency of PSCI ranges widely from 10-82% [2,3]. PSCI is associated with impairment of activities of daily living, functional disability, and increased risk of mortality [4,5]. It may also cause ischemic stroke recurrence [6]. However, the pathogenesis of PSCI remains unclear. In addition, precise biomarkers that could improve the prediction of cognitive impairment after ischemic stroke are still lacking.
Previous studies reported that the alternation and overexpression of periostin was found in a variety of diseases including ischemic stroke and other cerebrovascular diseases [10][11][12]. Recently, a cohort study of 162 large artery atherosclerotic stroke patients showed that serum periostin levels were positively correlated to the national institutes of health stroke scale (NIHSS) score and stroke volume [10]. Also, a growing body of evidences indicated that periostin is involved in the neuropathological processes, such as in ammation, disruption of blood-brain barrier, and neuronal cell death [13,14], which may lead to cognitive impairment. To date, the relationship between cognitive function status and periostin levels has not been clari ed yet. Therefore, we conducted this prospectively study to investigate whether the serum perostin levels at admission are associated with PSCI at 3 months.

Study design and patients
This observational prospective cohort study screened consecutive acute ischemic stroke patients hospitalized in The Sixth People's Hospital of Chengdu, between January 2019 and January 2020.
Patients were recruited in this study if they: (1) aged 18 years or old; (2) hospitalized within 7 days of symptoms onset; (3) had severe motor and language disabilities that precluding the cognitive evaluation.
Exclusion criteria for this study were a history of traumatic brain injury, drug and alcohol dependence, Parkinson disease, Alzheimer's disease, psychiatric disorders known to in uence cognitive function, and lost to follow up. This study was approved by the ethics committee at The Sixth People's Hospital of Chengdu, and all participating patients had provided informed consent before entering the study. The study was carried out according to the tenets of the Declaration of Helsinki.

Data collection
Demographic data, educational status, and vascular risk factors (including hypertension, diabetes mellitus, hyperlipidemia, coronary heart disease, previous stroke, and current smoking and drinking) were recorded at admission. We also collected body mass index, blood pressure, stroke severity and stroke etiology [15]. Baseline stroke severity was assessed by certi ed neurologist using NIHSS score [16]. Stroke subtype was classi ed using Trial of Org 10172 in Acute Stroke Treatment criteria [17].

Laboratory testing
Blood samples were obtained from each subject within 24 h after admission. The specimens were centrifuged at 1500 g for 15 min and the isolated serum frozen at − 80°C for further analysis. Serum periostin levels were measured using a commercially available ELISA kit (BioVendor, RAG019R). Intraassay and inter-assay coe cients of variation were < 10.0% and < 12.0%, respectively. All procedures were performed in strict accordance to manufacturers' instructions by laboratory technicians blinded to the clinical characteristics and outcomes. The other laboratory data including fasting blood glucose, lowdensity lipoprotein and Hyper-sensitive C-reactive protein (Hs-CRP) were also recorded.

Statistical Analysis
All statistical analysis was done with the SPSS software, version 24.0 (IBM, New York, NY) and R statistical software version 3.6.2. Categorical variables were expressed as percentages and analyzed with the chi-square test or Fisher's exact test. Continuous variables were summarized as mean (standard deviations) or medians (interquartile ranges) and compared by Student's t test, Mann-Whitney U test, analysis of variance or Kruskal-Wallis test [18]. We conducted 2 multiple adjusted logistic regression models to estimate the association of periostin levels with PSCI. Model 1 adjusted for age, and sex.
Model 2 included age, sex and covariates with a P value < 0.1 in the univariate analysis (including educational years, hypertension, diabetes mellitus, and NIHSS score). Restricted cubic spline regression was used to detect the shape of association between periostin and PSCI, tting a restricted cubic spline function with four knots (at the 5th, 35th, 65th, and 95th percentiles) [18]. We further performed subgroup analyses and investigated the potential modi ed effect of 6 interesting factors on the association between serum periostin and PSCI. Interactions between periostin and subgroup variables on the PSCI were tested by the likelihood ratio test with adjustment for the aforementioned covariates unless the variable was used as a subgroup variable. In all analyses, a P value < 0.05 was considered statistically signi cant.

Results
In our study cohort, a total of 315 consecutively admitted patients (mean age, 66.8 ± 8.9 years; 53.3% male) with acute ischemic stroke met the entry criteria. The median periostin levels were 17.6 ng/mL (interquartile range 14.2-20.7 ng/mL). Baseline epidemiological and clinical characteristics of the study population strati ed by the quartile of serum periostin concentrations were demonstrated in Table 1.
Subjects with elevated serum periostin levels were more likely to have a higher Hs-CRP levels (P = 0.038) and NIHSS score (P = 0.021). During the 3-month follow-up, 76 participants (24.1%) had mild PSCI and 97 (30.8%) had severe PSCI. Serum periostin levels were signi cantly higher in patients with severe PSCI than in those without PSCI (P for trend = 0.001) (Fig. 1). Patients in the PSCI group had less education (69.4% versus 57.0%; P = 0.024), a higher NIHSS score (median 9.0 versus 7.0; P = 0.001), and higher prevalence of hypertension (65.9% versus 48.6%; P = 0.002) as well as diabetes mellitus (31.8% versus 20.4%; P = 0.023) ( Table 2).  In subgroup analyses strati ed by age, gender, hypertension, diabetes mellitus, educational status, and stroke severity, the positive relationships between serum periostin concentrations and the risk of 3-month PSCI were found in all subgroups and reached statistically signi cance. Furthermore, no signi cant interaction between periostin levels and these interesting factors on the PSCI was observed (all P for interaction > 0.05; Fig. 3).

Discussion
Our prospective study recruited a group of acute ischemic patients and investigated relationship between serum periostin concentrations and cognitive function status. We found that higher serum periostin levels were associated with an increased risk of PSCI at 3 months. Furthermore, this association was independent for demographic characteristics, vascular risk factors and stroke severity. Hence, periostin in serum might represent a novel prognostic biomarker of PSCI.
Reported frequency of PSCI is ranges widely from 10-82% [2,3]. The discrepancy of PSCI incidence may be mainly attributed to differences in race, age, educational status, diagnostic criteria, and study methods. Most studies have assessed cognitive function at 3 months after a stroke using cross-sectional design [21][22][23][24][25], whereas some studies have adopted longitudinal designs [3,26]. Several diagnostic tools have been applied to investigate the presence of PSCI, such as National Institute of Neurological Disorders and Stroke and Canadian Stroke Network (NINDS-CSN), Mini-Mental State Examination (MMSE), and MoCA. In studies performed among populations of white dominance, the prevalence of 3month cognitive impairment after ischemic stroke ranges from 24-39% according to the MMSE, while the prevalence in the same population is up to 96% using a battery of neuropsychological tests [24,26]. However, in studies using a total MoCA score < 26 as the de nition of PSCI, the prevalence of PSCI approximates to 50% [22,23]. In the present study, we adopted a total MoCA score < 25 as the de nition of PSCI because this cutoff had a good sensitivity (77%) and speci city (83%) for mild cognitive impairment [19]. We observed that PSCI occurred in 54.9% of patients, which is similar to the ndings of other observational studies that have used the same diagnostic criteria [20,24].
Periostin is a secreted extracellular matrix protein that plays an important role in tissue repair, oncology, cardiovascular and central nervous systems, and in various in ammatory settings and diseases [7]. Previous studies found that increased periostin levels were associated with clinical severity and poor prognosis in patients with ischemic stroke [10], intracerebral hemorrhage [11], and aneurysmal subarachnoid hemorrhage [12]. Our study extended the current knowledge about the role of periostin in cerebrovascular diseases as it con rmed a positive relationship between circulating periostin concentrations and cognitive impairment after stroke. Both of clinical studies showed that periostin may play a crucial role in pathologic process after brain tissue injury.
According to the results from animal experiment, periostin 2 (splicing variant of periostin) was overexpressed at 24 hours after transient middle cerebral artery occlusion model. Exogenously injection of periostin 2 could reduce infarct volume [13]. Periostin has also been reported to promote neural stem cell proliferation and differentiation after hypoxic-ischemic injury. Intracerebroventricular administration of periostin was shown to signi cantly improve spatial learning and memory, indicating that periostin may alleviate cognitive de cits [27]. Periostin continued to be expressed up to 4 weeks after cerebral ischemia in various cells, such as astrocytes, microglia, and neuronal progenitor cells [14]. Therefore, the increase in circulating periostin levels might occur to repair the brain tissue and improve cognitive function after ischemic stroke. On the contrary, other studies demonstrated a detrimental effect of periostin [28,29]. Anti-periostin antibody improved post-subarachnoid hemorrhage neurobehavior, brain edema, and blood-brain barrier disruption. Recombinant-periostin signi cantly aggravated early brain injury [28]. Furthermore, the inhibition of periostin expression may improve cardiac systolic ejection function and animal survival rate [29]. Thus, researchers have not reached a consensus on the net effect of periostin. However, this mechanism warrants to be veri ed in future studies. Further studies are needed to detect the precise mechanisms underlying the association between elevated circulating periostin concentrations and cognitive impairment after ischemic stroke.
There were several limitations that should be addressed in this study. First, all 315 ischemic stroke patients in our study were prospective recruited from a tertiary referral hospital, so it is di cult to generalize from the results of this study. Second, patients with a history of cognitive impairment or a severe condition after stroke, whowere unable to complete psychological assessment, were excluded from the study. This selection bias would probably reduce the power of study. Third, serum periostin levels were only measured at one time point after admission, which may lead to some misclassi cation of exposure. Furthermore, multiple factors may in uence the accuracy of a single neuropsychological test in the diagnosis of PSCI, including educational status, physiological condition, and sensitivity or speci city of the test itself. The combined application of other evaluation tests in addition to MoCA, such as NINDS-CSN and MMSE, could improve the accuracy of the diagnosis. Therefore, the interpretation of our results should be cautious, and further multi-center studies with larger sample sizes are needed to validate our ndings.
In summary, our study provided preliminary data showing that increased serum perostin concentrations at baseline were independently associated with increased risk of PSCI among ischemic stroke patients. Further preclinical studies are warranted to investigate the underlying pathogenesis as well as targeted interventions in PSCI prevention and treatment.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee at The Sixth People's Hospital of Chengdu, and all participating patients had provided informed consent before entering the study. The study was carried out according to the tenets of the Declaration of Helsinki.

Consent for publication
Not applicable.

Availability of data and materials
The data that support the ndings of this study are available on request from the corresponding author.

Competing interests
All the authors declare that there is no con ict of interest.

Funding
None.
Authors' contributions GZL, ML conceived and coordinated the study, designed, performed and analyzed the experiments, wrote the paper. JL, CZ, JY, BW, XJL carried out the data collection, data analysis, and revised the paper. ML designed the study, carried out the data analysis, and revised the paper. All authors reviewed the results and approved the nal version of the manuscript.  Figure 1 Comparison of serum periostin levels and cognitive impairment severity at 3 months.

Figure 2
Restricted cubic spline regression assessed the association between serum periostin and risk of poststroke cognitive impairment. Odds ratios and 95% con dence intervals derived from restricted cubic spline regression, with knots placed at the 5th, 35th, 65th, and 95th percentiles of the periostin levels. The reference point for serum periostin is the midpoint (17.6 ng/mL) of the reference group from categorical analysis. Odds ratios were adjusted for the same variables as model 2 in Table 3.