Serum β-synuclein, neurofilament light chain and glial fibrillary acidic protein as prognostic biomarkers in moderate-to-severe acute ischemic stroke

We aimed to assess the prognostic value of serum β-synuclein (β-syn), neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in patients with moderate-to-severe acute ischemic stroke. We measured β-syn, GFAP and NfL in serum samples collected one day after admission in 30 adult patients with moderate-to-severe ischemic stroke due to middle cerebral artery (MCA) occlusion. We tested the associations between biomarker levels and clinical and radiological scores (National Institute of Health Stroke Scale scores, NIHSS, and Alberta Stroke Program Early CT Score, ASPECTS), as well as measures of functional outcome (modified Rankin Scale, mRS). Serum biomarkers were significantly associated with ASPECTS values (β-syn p = 0.0011, GFAP p = 0.0002) but not with NIHSS scores at admission. Patients who received mechanical thrombectomy and intravenous thrombolysis showed lower β-syn (p = 0.029) und NfL concentrations (p = 0.0024) compared to patients who received only mechanical thrombectomy. According to median biomarker levels, patients with high β-syn, NfL or GFAP levels showed, after therapy, lower clinical improvement (i.e., lower 24-h NIHSS change), higher NIHSS scores during hospitalization and higher mRS scores at 3-month follow-up. Elevated serum concentrations of β-syn (p = 0.016), NfL (p = 0.020) or GFAP (p = 0.010) were significantly associated with 3-month mRS of 3–6 vs. 0–2 even after accounting for age, sex and renal function. In patients with moderate-to-severe acute ischemic stroke, serum β-syn, NfL and GFAP levels associated with clinical and radiological scores at different timepoints and were able to predict short- and middle-term clinical outcomes.

www.nature.com/scientificreports/In recent years, blood-based biomarkers have been largely investigated to better prognosticate patients with acute ischemic stroke 1 .Among the most promising ones, neurofilament light chain protein (NfL) and glial fibrillary acidic protein (GFAP) represent surrogates of neuroaxonal and glial injury, respectively 2,3 .In previous studies on minor-to-moderate ischemic stroke, NfL and GFAP showed a fair value as prognostic factors to predict short-and middle-term functional outcomes as well as all-cause mortality [2][3][4] .However, the role of such markers in patients with severe ischemic stroke has been only partly investigated 5 .In addition, β-synuclein (β-syn) is emerging as a biomarker for synaptic damage in several neurological disorders, such as neurodegenerative [6][7][8][9] and traumatic diseases 10 .The promising results obtained so far encourage the assessment of blood β-syn also in cerebrovascular diseases, where its profile is, to date, still unexplored.These facts considered, the present exploratory analysis of a prospective single-center cohort aimed to test the possible associations of serum β-syn, NfL and GFAP levels with clinical and radiological scores and with measures of functional outcome in patients with moderate-to-severe acute ischemic stroke.

Inclusion criteria
We included patients with acute ischemic stroke due to middle cerebral artery (MCA) occlusion in the M1 segment, enrolled into a single-center perspective cohort (DRKS00022064) at the University of Würzburg (Germany) between 07/2020 and 08/2021 (detailed patients selection from the main cohort in Supplementary Fig. S1), which has been partly published elsewhere 11 .Inclusion criteria were a clear time window from clinical onset and an admission National Institute of Health Stroke Scale (NIHSS) score ≥ 6 and/or acute treatment with mechanical thrombectomy (MT).From all patients (n = 30), we collected baseline data on the Alberta Stroke Program CT Score (ASPECTS) and NIHSS scores.After treatment (described in detail in the "Results" section), we collected data on: ASPECTS after 24-72 h 12,13 ; NIHSS scores after 24, 48 and 72 h and at discharge; modified Rankin Scale (mRS) at discharge and at 3-month follow-up.

Blood sampling and analysis
Blood samples were collected one day after admission at scheduled timepoints (10am) (median time from clinical onset: 22 h 40 m; interquartile range, IQR 18 h 45 m-23 h 04 m) and analyzed according to standard procedures.We measured NfL by using a commercial kit for the Ella microfluidic system (BioTechne, Minneapolis, USA), GFAP with a commercial Simoa kit on a HD-X platform (Quanterix Inc., Lexington, USA) and β-syn with our recently described in-house established digital immunoassay 14 .In all measurements, the intra-and inter-assay coefficients of variability were < 10% and < 15%, respectively.

Statistical analysis
Statistical analysis was performed with GraphPad 8 (GraphPad Software, La Jolla, USA) and R v4.2.2 (R Foundation, Vienna, Austria).We used the χ 2 test and the Mann-Whitney test for two-group comparisons of categorical and continuous variables, respectively.For comparisons among multiple groups, we adopted the Kruskal-Wallis test.We calculated correlations between biomarker levels with the Spearman's coefficient.Linear regression models were used to test associations between baseline characteristics and biomarker levels, as well as between biomarker levels and continuous clinical outcomes.Logistic regression models were adopted to test associations of serum biomarkers with binary outcomes 12,13,15 (see section "Definition of binary outcome measures").Multivariable analyses were adjusted for age, sex and creatinine levels.Nagelkerke's R 2 was used to estimate the explained variance of regression models.To test the diagnostic accuracy of biomarkers, we performed receiver operating characteristic (ROC) analysis.Biomarker concentrations were log-transformed for all analyses and p-values < 0.05 were considered statistically significant.

Study protocol approval
This study was conducted in accordance with the Helsinki Declaration and its recent modifications.All patients gave written informed consent to participate in the research and the local Ethics Committee of the University of Würzburg approved the study protocol (reference n. 05/20-am).

Baseline characteristics of the study population
In our study cohort, patients had a mean age of 75.1 (± standard deviation: 11.3) years and 17 participants (56.7%) were female.At admission, patients had median ASPECTS values of 7 (interquartile range, IQR 7-8) and median NIHSS scores of 14 (IQR 10-17).Patients were admitted after (median and IQR) 3 h 11 m (2 h 18 m-4 h 07 m) and blood samples were collected after (median and IQR) 22 h 40 m (18 h 45 m-27 h 04 m) from clinical onset.The Trial of Org 10172 in Acute Stroke Treatment (TOAST) classification system 16 was used to distinguish different stroke etiologies, which were large-artery atherosclerosis in 5 cases, cardioembolism in 15 cases, other determined etiology in 4 cases and undetermined etiology in 6 cases.Moreover, 5 patients had www.nature.com/scientificreports/previous neurological disorders in their medical history (dementia n = 1, Parkinson's disease n = 1, previous stroke n = 1, delirium n = 2).Further clinical and biochemical data are reported in Table 1.

Clinical and neuroradiological progression according to median biomarker levels
After stratification of patients according to median β-syn (20.1 pg/ml), NfL (50.4 pg/ml) and GFAP (6.3 ng/ml) concentrations, patients with higher biomarker levels (n = 15) had lower ASPECTS values and higher NIHSS scores after therapy than patients with low biomarker concentrations (n = 15), even though they did not differ   www.nature.com/scientificreports/ in their admission NIHSS scores (Fig. 1D-F, Supplementary Table 1).Moreover, the former group experienced an overall lower clinical improvement soon after therapy (i.e., lower 24-h ΔNIHSS) compared to the latter group (Fig. 1G).Interestingly, we also observed significantly lower mRS scores in patients with high vs. low β-syn, NfL and GFAP levels both at discharge and at 3-month follow-up (Supplementary Table 1).

Associations between serum biomarkers and clinical outcomes
During the hospitalization period, 5 patients died because of severe neurological complications (malignant MCA infarction n = 3, hemorrhagic infarction n = 2) and 3 patients died because of severe non-neurological www.nature.com/scientificreports/complications (i.e., aspiration pneumonia).Two additional patients died because of severe aspiration pneumonia before 3-month follow-up.In our cohort, only NfL levels were higher in non-survivors (n = 8) vs. survivors (n = 22, p = 0.031) (Fig. 1H).After accounting for the cause of death, patients who died because of severe neurological complications (n = 5) had significantly higher levels of β-syn (p = 0.027) and NfL (p = 0.019) compared to all other patients (n = 25), but not compared only to patients who died because of aspiration pneumonia (n = 5) (Supplementary Fig. 2).Moreover, Both β-syn and NfL were significantly positively associated with the risk of death due to severe neurological complications at univariate but not at multivariate logistic regression analysis (Supplementary Table 2).Finally, patients with 3-month mRS of 3-6 showed elevated serum β-syn (p = 0.0023), NfL (p = 0.0011) and GFAP (p = 0.0045) concentrations compared to patients with 3-month mRS of 0-2 (Fig. 1I).Similar results were found when considering the mRS scores at discharge (Supplementary Fig. 2).Of notice, all three serum biomarkers were significant predictors of unfavourable outcome at 3-month follow-up both at uni-and at multivariate analysis after accounting for age, sex and renal function (Supplementary Table 2).

Discussion
In the present study, we reported on the prognostic value of blood β-syn, NfL and GFAP in patients with moderate-to-severe acute ischemic stroke.Our preliminary data showed that β-syn and GFAP concentrations measured at day 1 after reperfusion were significantly associated with admission and post-interventional ASPECTS, suggesting a direct relationship between the biomarker levels and the extent of the ischemic injury.Instead, we found no associations between NfL and ASPECTS values at admission, which is in line with previous studies that reported associations with infarct volume when the biomarker was measured after 3 to 7 days from the ischemic event but not within 24 h 4,17 .Furthermore, the lack of significant associations between β-syn, NfL or GFAP concentrations and baseline NIHSS score could be due to the lack of a strict relationship between NIHSS scores and ASPECTS 18 .Alternatively, such associations might be detectable only in patients with minor stroke 17 .
On another issue, it is still poorly explored what impact different types of acute treatment (i.e., MT and IVT alone or in combination) may have on brain-specific biomarkers 5,19,20 .Our preliminary results suggest that the use of IVT in addition to mechanical revascularization may be associated with lower biomarker levels compared to patients who received only MT, but larger confirmative studies are needed.Most interestingly, our findings suggest a possible role of serum biomarkers for monitoring clinical progression at different timepoints after therapy, both during hospitalization and at follow-up.We could largely confirm previous results on the good prognostic value of blood NfL and GFAP in minor-to-moderate acute ischemic stroke [2][3][4] , but also highlight the potential utility of such biomarkers for severely affected patients 5 .Indeed, serum NfL seemed to be more associated with all-cause mortality, in line with previous evidence in healthy elderlies 21 .Instead, GFAP blood concentrations may change with a rapid dynamics within few hours after stroke and acute treatment 5,10 , suggesting that its prognostic value might be strictly time-dependent.Hence, blood-based biomarkers with different temporal patterns might be used in combination for prognostication during distinct disease stages.
As a strength of our work, we included in the analysis factors that are well-known in literature for influencing serum biomarker levels, such as age and renal function 22 .On the other hand, the small sample size represents the major limitation of our exploratory single-center study and multiple testing in such sample might increase the risk of false-positive results.Moreover, blood collection was performed only at a single timepoint during the acute post-treatment phase.Other studies showed that the performance of some biomarkers (e.g., NfL) for predicting clinical outcomes in patients with ischemic stroke may be better when measured, for example, after 7 days from the event 3 .Instead, the temporal dynamics of GFAP and β-syn in cerebrovascular diseases, as well as their possible utility when measured at later timepoints, remains still unexplored.Moreover, we observed in our cohort a higher proportion of patients who died because of aspiration pneumonia compared to previous data from a recent meta-analysis 23 , which could be due to an older age at onset and a higher disease severity.To date, the prognostic value of blood biomarkers in relation to different causes of death has not been assessed yet.Hence, our preliminary results should be replicated in independent and larger cohorts to address these missing points.

Conclusions
In conclusion, serum β-syn levels were associated with clinical and radiological scores of stroke severity and with measures of functional outcome in this exploratory analysis.Moreover, NfL and GFAP showed distinct prognostic utility.Further studies on larger cohorts should better test the possible additive prognostic role of β-syn if compared to NfL and GFAP.

Table 1 .
Clinical, radiological and biochemical data of the study population.Age is reported as mean ± standard deviation, other continuous variables as median (interquartile range).*1 dementia, 1 Parkinson's disease, 1 previous stroke, 2 delirium.