Circulating neurofilament light in ischemic stroke: temporal profile and outcome prediction

Serum neurofilament light chain (sNfL) has recently been suggested as a marker of neuroaxonal injury after ischemic stroke with potential applications both for patient monitoring and for observational and interventional studies [1]. NfL is a neuron-specific structural protein [2] and cerebrospinal fluid concentrations can be used as markers of neuronal damage [3]. The recent development of methods to quantify NfL in serum has led to several reports on sNfL in neurodegenerative disorders and traumatic brain injury [4‐6] and opens the door for large studies on NfL as a marker of neuronal damage also in diseases where lumbar puncture is not part of the clinical routine, such as stroke. However, data on sNfL in ischemic stroke are still limited to a few published studies [1, 7‐9]. Consistent with what would be expected of a marker of neuronal damage, results from these studies show higher sNfL in patients with acute ischemic stroke compared to both patients with a recent transient ischemic attack [8, 9] and controls [1, 7], and correlations between sNfL and infarct volume have been observed [1, 7]. There is evidence that sNfL increases with the time from symptom onset to blood-draw [1, 7, 8], and that sNfL remains elevated at 3 months post-stroke [1, 7]. Furthermore, recent data indicate that sNfL measured at day 7 after symptom onset, but not as early as within 24 h, is independently associated with 3-month functional outcome [1, 9], illustrating that the time point of measurement is of great importance when evaluating sNfL post-stroke.

Although reported data show promising results for sNfL as a biomarker in stroke, the post-stroke temporal profile and prognostic value remain to be evaluated in large samples also enabling analyses of relevant subgroups. In light of this, we aimed to explore sNfL in ischemic stroke in a longitudinal manner and to study associations for sNfL at different time points after ischemic stroke to both functional and neurological outcomes. We hypothesized that sNfL measured in the subacute or convalescent phase predicts post-stroke outcomes, and that the concentrations and/or the time profile of sNfL differ between etiologic subtypes.

In five patients serum was unavailable from all time points, leading to a study cohort of 595 patients with serum sample available from at least one time point and their corresponding matched controls. Baseline characteristics and outcomes for the whole group and for the four main etiological stroke subtypes are shown in Table 1. As expected, the proportion of participants with cardiovascular risk factors and cardiovascular comorbidities was higher in cases than in controls. In cases, there were correlations between sNfL and stroke severity (r = 0.38 and r = 0.56 for acute phase and 3-month sNfL, respectively). sNfL was also correlated to age in controls (r = 0.50), whereas all other correlations between sNfL and baseline characteristics were weak, |r| ≤ 0.2. Baseline characteristics for the subgroup who participated in the 7-year follow-up are shown in Supplemental Table 1 (online resource 1).

In this cohort of ischemic stroke, circulating sNfL concentrations increased with the day of blood draw in the acute phase after index stroke, were highest at 3 months, and had declined 7 years post-stroke. sNfL at 3 months post-stroke showed strongest independent associations to both short- and long-term outcomes. Finally, we observed that the dynamics of sNfL differ by subtype of ischemic stroke.

For the first time we show that sNfL predicts both functional and neurological outcomes both in the short- and long-term after overall ischemic stroke. In our cohort, associations between acute phase and 3-month sNfL and outcomes were independent of age and stroke severity, which is in line with a recent but smaller study (n = 110) with data on sNfL measured at day 7 from admission [1]. In contrast, no independent association between sNfL and 3-month mRS was detected when blood was drawn within 24 h from symptom onset [9]. Thus, according to the present and previous studies [7, 8], 24 h is too early to catch the sNfL peak. In a study on non-traumatic cervical artery dissection [8], blood was drawn at similar time points as the acute phase sampling in the present study, but an association between sNfL and 3-month mRS did not remain after adjustment for stroke severity [8], which could be due to the small sample size (n = 49) and the fact that the electrochemiluminescence immunoassay was used to measure sNfL, which is less sensitive compared to the method we used here [16]. We used the ultrasensitive SiMoA technology to measure sNfL levels longitudinally after stroke, and for the first time these were investigated in relation to outcomes measured by both NIHSS and mRS scores at different time points. These results need replication, but indicate that sNfL is a promising candidate for prediction of short- and long-term outcomes after ischemic stroke. In contrast to most previously studied biomarkers of stroke outcomes that have mainly focused on inflammatory and hormonal pathways, the neurofilament proteins are exclusively expressed in neurons and thereby represent markers specific for neuroaxonal injury. This means they are independent of the underlying causal pathways and provide a measure of brain injury with the advantage of being much more readily available compared to brain imaging techniques. However, as indicated above the time point of measurement seems to be crucial when evaluating sNfL post-stroke. The warranted next step is to establish the post-stroke temporal profile of sNfL. As discussed below, the peak is probably not fully captured in our or previous studies, and speculatively measurements of peak levels could add predictive ability of sNfL. However, repeated measurements might be even more valuable, by measuring the rates of increase and decrease in sNfL and, thus capturing the dynamics of post-stroke injury processes.

We observed significantly higher sNfL in ischemic stroke cases compared to controls in the acute phase, after 3 months and 7 years, and this was also true when excluding participants with neurological comorbidities. After 7 years the sNfL had declined, and the association to case–control status was not independent of age and cardiovascular risk factors/comorbidities. The magnitude of the sNfL increment within 3 months post-stroke was associated to stroke severity, i.e., baseline NIHSS, which is in line with previous data [8, 9] and expected since both are measures related to the magnitude of brain injury. In line with this, sNfL differed between the OCSP subtypes with lowest acute phase levels in LACI. We did not have data on infarct size and localization, but speculate that sNfL and the NIHSS score represent overlapping but complementary measures of the magnitude and functional consequences of brain damage. Furthermore, our results indicate that sNfL continues to increase several days after stroke. Similar associations between time of blood draw and sNfL have been reported in lacunar stroke [7], ischemic stroke caused by cervical artery dissection [8], and traumatic spinal cord injury [17]. Moreover, a recent study on a subgroup of patients with acute ischemic stroke (n = 89) who had measurements of sNfL on day 1, 2, 3, and 7 after hospital admission observed highest concentrations at day 7 [1] and a study on phosphorylated neurofilament heavy protein observed highest concentrations at 3 weeks post-stroke [18]. Interestingly, a small recent study on 30 ischemic stroke patients with repeated measurements of sNfL on day 0–1, 2–3, 7–9, 3 weeks and 3–5 months confirmed these findings with increasing sNfL during the acute phase and highest concentrations at 3 weeks [19]. In our study, sNfL 3 months post-stroke was significantly higher compared to the acute phase, but contrary to the acute phase, the 3-months concentrations were decreasing with the day of blood-draw. In addition, as depicted in Supplemental Fig. 1 (online resource 1), a larger proportion of patients with acute phase blood sampling ≤ 4 days post-stroke had higher sNfL at 3 months compared to patients with acute blood sampling later than 4 days post-stroke. Taken together, these data point towards a peak in sNfL somewhere between the acute phase and 3 months post-stroke. The half-life of NfL in humans is not known, but data from mice have suggested a half-life around 3 weeks [20]. Neurofilaments are abundant in large myelinated axons susceptible to Wallerian post-stroke degeneration and the time profile of immunohistochemical as well as radiological findings after stroke show a delayed and longstanding degenerative process fitting the release pattern of sNfL in this study [21, 22]. Thus, the prolonged release of NfL possibly reflects these processes. This is further supported by recent data showing a correlation between sNfL measured 6 months post-stroke and a quantitative measure of secondary neurodegeneration obtained from diffusion tensor imaging MRI [1]. Although we do not know when sNfL is peaking after stroke, it is reasonable to assume that this time point may vary with infarct size. In support of such a hypothesis, in contrast to our results on all ischemic stroke and previous on lacunar stroke [7], sNfL in amateur boxers had decreased after a 3-month period of rest from boxing [5], which could be due to a milder brain injury compared to stroke. Taken together, present data on the temporal profile of sNfL post-stroke are not sufficient to identify the post-stroke peak. Therefore, we cannot draw any conclusions on the optimal time(s) for measurement, but clearly the very first days after stroke are too early.

We found that the sNfL profile differs by etiologic subtype of ischemic stroke. LVD and CE stroke showed highest acute phase and 3-month concentrations, which is expected since those etiologies in general cause larger strokes. The median 3-month sNfL concentrations for LVD were 190 pg/mL, CE stroke 136 pg/mL and SVD 56 pg/mL. For comparison, reported sNfL concentrations were approximately 30 pg/mL in multiple sclerosis [6], 80 pg/mL in frontotemporal dementia [4], and 22 pg/mL in mild traumatic brain injury (amateur boxers sampled 7–10 days after boxing) [5]. It should be noted, however, that these comparisons are only indicative; the assays used in these studies are similar (the same antibody pair to measure NfL was used) but have not been formally standardized to a reference material or harmonized to each other. Given the promising results on NfL as a blood biomarker for neuronal injury, such standardization projects are highly warranted. At 7 years, SVD showed highest sNfL, although at this time point the difference between the subtypes was not statistically significant in our sample. It is plausible that the concentrations are constantly elevated in some patients with SVD, due to a progressive disease course. In line with this, previous studies on NfL in cerebrospinal fluid have demonstrated increased concentrations with increasing severity of white-matter lesions on magnetic resonance imaging (MRI) [23, 24]. Moreover, a study on patients with small subcortical infarcts (< 25 mm) showed elevated sNfL in patients with new, but clinically silent cerebral small vessel disease-related lesions on MRI at follow-up [7]. This finding was further supported by a recent study showing that ischemic stroke patients with recurrent ischemic lesions on MRI at 6 months had higher sNfL compared to those without new lesions [1].

Our study has several strengths. The longitudinal design with repeated blood-draws and assessments of outcomes in short- and long-term provide novel information on the dynamics of sNfL, its relation to ischemic stroke subtypes, and its potential as a predictor of ischemic stroke outcomes. Further, we assessed two different outcome measures. Functional outcome as assessed by the mRS represents a crude measure, but the dichotomization refers to differences of great importance clinically and for the patients. The NIHSS assesses neurological outcome and is more detailed compared to the mRS. Our study sample was large and comprised of consecutive, well-characterized and relatively young ischemic stroke patients, which is why the proportion of comorbidities was low. Blood sampling was strictly standardized and sNfL was measured by the most sensitive method available [16]. Our study also has limitations. Although we had serum samples available from three time points, and those in the first phase after stroke were spread over a period of time, we did not have repeated measurements during the acute phase and no measurements between the acute phase and 3 months. Therefore, our data are insufficient to identify the post-stroke peak in sNfL. Blood sampling in narrower time intervals over the first weeks and months is needed in future studies to determine the trajectories of sNfL after stroke. Moreover, index stroke severity was scored as maximum severity within the first 7 days and it is possible that some cases, especially those with early blood sampling, have experienced a deterioration leading to falsely low sNfL concentrations in relation to stroke severity. Moreover, sNfL is known to associate with cerebral small vessel burden [25]. Since we did not have available data on small disease burden for all cases or any of the controls, we were not able to account for this in our analyses. There were also several years between blood sampling and sNfL analyses. However, all samples were stored at − 80 °C pending analysis, and NfL is known to be stable at − 80 °C and has also been shown to be stable over multiple freeze–thaw cycles [26]. Finally, our study cohort consisted of young and middle-aged ischemic stroke patients which may reduce the external validity of our results.

Patients with acute ischemic stroke have increased sNfL concentrations, and the dynamics of sNfL differ by stroke subtype. There is an association between sNfL and both neurological and functional outcomes, which is independent of stroke severity. The results from this study, which represent the largest analyses of sNfL after ischemic stroke and also include repeated measures of both sNfL and outcomes, improve our understanding of sNfL in ischemic stroke. Future studies should aim to establish the time profile of sNfL post-stroke to enable identification of the optimal time for measurements.