Stroke is still a major public health issue that shortens adults’ lives worldwide (Feigin et al., 2018). The majority of strokes are ischemic, and survivors are more likely to develop permanent disabilities (Diener and Hankey, 2020). Therefore, strategies to accurately risk assess the prognosis of ischemic stroke at the acute stage will be of great value in identifying patients at high risk of poor prognosis promptly and could offer an alternative approach to monitoring patients. Although imaging and conventional cerebrovascular risk factors are helpful in the diagnosis and intervention of ischemic stroke, they cannot fully explain the clinical process and predict outcomes after AIS onset (Guo et al., 2020). Therefore, novel blood biomarkers capable of indicating the likelihood of stroke progression and prognosis have gained interest in recent decades. Multiple interesting markers that are possible to involve neurovascular units (NVUs) impairment cascade after stroke have been identified, such as endothelial dysfunction markers, neural plasticity and vascular remodeling markers and inflammatory markers (Chen et al., 2012; Garcia-Rodriguez et al., 2021; Przykaza, 2021). Among them, inflammatory biomarkers have drawn the most interest, so far none of them have been approved for the clinical management of stroke.

Matrix metalloproteinase-9 (MMP-9) is one of the most extensively studied circulating biomarkers in ischemic stroke because of its role in neuroinflammation, early vasogenic edema, neuronal apoptosis, vascular remodeling, and neurorepair during the ischemic stroke pathobiological process (Vafadari et al., 2016; Montaner et al., 2019; Figiel et al., 2021). Some observations found that higher circulating MMP-9 levels were associated with neurological deficit, infarct volume (Montaner et al., 2001; Morancho et al., 2010), and an increaserisk of poor prognosis (Ning et al., 2006; Guo et al., 2020), while others found no association between MMP-9 and worse outcomes (Tsuruoka et al., 2014; Maestrini et al., 2020). Brain-derived neurotrophic factor (BDNF), a neurotrophin coordinating neuroplasticity in neurological disorders (Farmer et al., 2004; Bramham and Messaoudi, 2005), is another promising blood biomarker that has been investigated in ischemic stroke. Despite the growing evidence for the involvement of BDNF and neuroinflammation in brain disorders (Lima Giacobbo et al., 2019), the association between the circulating level of BDNF and functional outcome after AIS is controversial. Some studies indicated that low serum BDNF levels were associated with large infarct volumes or poor neurological outcomes after stroke onset (Stanne et al., 2016; Qiao et al., 2017; Wang et al., 2017), whereas others did not (Luo et al., 2019; Mourão et al., 2019).

The discrepancy in the findings of the relationship between the circulating MMP-9/BDNF and clinical outcome may partly be due to most studies with a single-occasion measurement of the prognostic metrics, which prevents studying the longitudinal changes of MMP-9 and BDNF during the acute stage of ischemic stroke. The modulations of MMPs and neurotrophin are complex since they may be sensitive to different facets of pathophysiology and may change over time after AIS. Little is known about the temporal behavior of such biomarkers’ expression. The aim of the study was to evaluate dynamic changes in blood MMP-9 and BDNF levels in the acute stage and their association with 3-month prognosis in patients with AIS.

The present study demonstrated that circulating levels of MMP-9 and BDNF were significantly elevated in acute ischemic stroke and has investigated the sequential changes in these two biomarkers after stroke onset and their association with stroke prognosis regardless of whether they are combined with thrombolysis. In the present study, we clarified that elevation of the baseline MMP-9 serum level and its gradual upward trend overtime at the acute stage were associated with a 3-month poor prognosis in AIS patients. BDNF expression levels were found to be higher in the early stages of patients with a poor prognosis compared to those with a good prognosis, but gradually decreased in the later stages. Similarly, this phenomenon was much more significant in the non-thrombolysis group as well.

Over the recent decades, circulating MMP-9 and BDNF concentrations have been found to have potential value in stroke diagnosis and prognosis. However, the prognostic value of circulating expression of MMP-9 in AIS remains elusive. Consistent with our results, a persistent increase in MMP-9 has been reported in stroke (Montaner et al., 2001). Increased peripheral MMP-9 levels have also been reported to be implicated in hemorrhagic transformation after ischemic stroke (Rosell et al., 2008), ischemic stroke severity and infarct volume (Rosell et al., 2005; Demir et al., 2012), and poor prognosis of AIS (Ramos-Fernandez et al., 2011; Gori et al., 2017; Zhong et al., 2017). Nevertheless, the Biostroke study indicated plasma MMP-9 concentrations were not associated with stroke severity and worse outcome (Maestrini et al., 2020). Regarding BDNF as a blood biomarker, inconsistencies are also seen in clinical studies of AIS, which focused on its prognostic impact of adverse outcomes. In the present research, we observed that a substantial decrease in BDNF was involved in the poor outcome at 3 months. For instance, some studies reported that low baseline BDNF was significantly associated with poor functional outcomes at 3 months (Lasek-Bal et al., 2015; Wang et al., 2017), and larger infarct volumes in AIS (Qiao et al., 2017).

At present, researchers have several perspectives on the biological mechanisms underlying the association between these two markers and ischemic stroke. After ischemic brain injury, vascular endothelium impairment leads to release and activation of MMP-9 (Turner and Sharp, 2016). MMP-9 can promote extracellular matrix degradation and increase blood-brain barrier (BBB) permeability, especially early as a mediator of neuroinflammation and later involved in neuroplasticity and vascular remodeling (Lee et al., 2006; Barkho et al., 2008; Guo et al., 2020; Figiel et al., 2021). Experiments in animals have shown that MMP-9 was upregulated in areas of cerebral ischemia and that a breakdown of the blood-brain barrier led to brain swelling, hemorrhage, and neurotoxic cell death in the early stages of stroke (Wang et al., 2020). BDNF may be involved in neuroprotection due to its role in neuronal development, differentiation, and survival and its involvement in homeostatic interactions in neurovascular units (Pikula et al., 2013; Calabrese et al., 2014). Post-ischemic neurogenesis occurs may represent an endogenous repair mechanism for ischemic stroke (Jiang et al., 2016). Experimental studies have demonstrated that administering intravenous BDNF has been shown to facilitate neurogenesis and improve functional recovery after stroke (Schäbitz et al., 2007). In animal models, BDNF levels increased in the brain and circulation immediately after an ischemic incident (Mourão et al., 2019). It was also found that exogenous BDNF affects vasodilation through promoting prostacyclin biosynthesis and protects isolated cerebral artery walls from vasoconstriction-related injury and thrombosis (Santhanam et al., 2010). Accordingly, BDNF may reduce the severity of ischemic stroke through its neurotrophic effects and its vascular protective effects.

The modulation of MMP-9 and BDNF is complex since they may have a pleiotropic and biphasic nature with multiple roles that vary greatly depending on the stage of the AIS. Furthermore, little is known about whether peripheral MMP-9/BDNF variations mirror brain changes in AIS pathophysiology and thus guide clinical interventions. Our findings demonstrated that MMP-9 levels gradually increased from Day 1 to Day 5 in the poor prognosis group, while they gradually decreased in the good prognosis group. There was a significant difference in MMP-9 concentrations between patients with and without a good prognosis on Day 5 after admission, but not at baseline or on Day 2. This finding was consistent with the study of Tsuruoka et al. (2014) which has indicated that increased MMP-9 levels measured at the hyperacute stage (at admission and 24 h after admission) of AIS were not associated with the short-term poor prognosis. Our results suggested that a persistent increased level of circulating MMP-9 could reflect the brain tissue injury and cell death in the acute stage after ischemic stroke (Rempe et al., 2016), which is related to the adverse outcomes rather than baseline expression. Notably, this association might be attenuated by the thrombolytic treatment.

Similar to the previous study (Wang et al., 2017), the current study also demonstrated that a gradually decreasing serum BDNF level after stroke onset appears to be a risk factor for poor prognosis at 3 months. In particular, our study demonstrated the biphasic time course of BDNF changes in patients with different prognoses, with a significant increase in BDNF levels early (Day 1 time point) and a reverse change later (Day 5 time point) in patients with poor prognosis. This association was more significant in patients without thrombolysis. The underlying mechanism of such changes in BDNF expression may be explained as follows: (a) Massive production of circulating BDNF level in patients with poor prognosis in the initial stage may implicate that the excess BDNF found in circulation comes from an alternative, but still speculative, source of peripheral endothelium rather than the brain. Endothelial cells synthesize and release large amounts of BDNF when exposed to oxidative stress and pro-inflammatory cytokines (Béjot et al., 2011a,b). The circulating BDNF at the initial stage after stroke onset may reflect the extent of oxidative stress and pro-inflammatory damage; (b) When the tissue damage is extensive, affecting the affecting the brain’s ability to produce BDNF, circulating decreased BDNF levels may reflect impaired neurogenesis and synaptic plasticity at a later stage of AIS (Calabrese et al., 2014). A previous study also reported that BDNF levels at 72 h of hospitalization but not at admission were associated with hospitalization days in AIS (Mourão et al., 2019). Accordingly, instead of circulating levels of BDNF in the first days after stroke, the sustaining reduction of peripheral BDNF expression at the acute stage is likely to correlate with poor clinical outcomes in patients with AIS.

The strengths of this study lie in the fact that the longitudinal changes of MMP-9 and BDNF are time-sensitive and may be more informative and promising in predicting patients’ prognosis. In addition, our results have indicated that thrombolytic therapy could affect the predictive value of MMP-9/BDNF in AIS, partly due to the fact that thrombolysis therapy may affect the expression of MMP-9 (Saleem et al., 2021). However, the results of our study should be interpreted in light of its limitations. First, the small sample size raises the possibility of undetected bias and does not allow adjustment for all possible confounders, including stroke etiology. In addition, the limited number of patients with thrombolysis may prevent us from examining group differences. Secondly, its single-center, retrospective design may result in selection bias. Although we have investigated the temporal profile of MMP-9 and BDNF concentrations, we emphasize that our results require confirmation in larger sample size and different stroke subtype populations. Furthermore, besides thrombolysis, other pharmacological or non-pharmacological interventions may affect changes in MMP-9 and BDNF. In the current study, we cannot perform an analysis of the impact of such interventions on changes in two biomarkers. Further studies are needed to determine these interventions in the context of their effect on ischemic stroke biomarkers.

In conclusion, MMP-9 and BDNF are closely related to the prognosis of patients with AIS in a time-dependent manner. In the initial stages of AIS, lower serum BNDF levels may predict patients with a favorable prognosis. The decrease in MMP-9 and increase in BDNF levels in the patient after several days of treatment implied a favorable prognosis. In clinical practice, dynamic changes of MMP-9 and BDNF were superior to baseline measurement in predicting the prognosis of ischemic stroke.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Institutional Review Board of The First People’s Hospital of Zhaoqing (B2021-12-01). The patients/participants provided their written informed consent to participate in this study.

YL and XHa: study concept and design, draft the manuscript, and funding acquisition. SL and HH: acquisition, analysis, and interpretation of the data. XHu and ML: administer the project. YH: supervise the study. YC and ZW: acquisition data and patients’ recruitment. All authors critically revised the manuscript, had full access to all the data in the study, and have read and approved the submitted version.