Intravenous alteplase is the main effective treatment for acute ischemic stroke (AIS) within 4.5 h after the onset of symptoms (1). Symptomatic intracranial hemorrhage (sICH) is a rare but severe complication after thrombolysis that is closely related to disability and death (2).

Mountains of studies have investigated predictors for post-thrombolytic sICH in stroke, such as clinical, radiological, and laboratory factors (3–5). Although several biomarkers were found to be associated with post-thrombolytic hemorrhagic transformation (6–11), these biomarkers were only detected at admission. To date, however, only one study investigated the change of serum biomarkers after post-thrombolytic sICH (12). Given that changes in biomarkers before and after sICH maybe reflect the secondary brain injury of sICH, the issue needed to be further investigated.

In the INtravenous Thrombolysis REgistry for Chinese Ischemic Stroke within 4.5 h of onset (INTRECIS) (13), 5 centers were pre-designed to consecutively collect blood samples before and 24 h after thrombolysis. In this study, we tried to identify what serum biomarkers change significantly before thrombolysis vs. after sICH, compared with patients without any ICH and investigated the potential interactions by microarray analysis on 49 preset biomarkers.

As shown in Figure 1, 358 patients with thrombolysis were consecutively screened in the present study, and 234 patients were excluded for different reasons: 160 patients received urokinase or the non-standard dose of alteplase, 10 patients received the endovascular intervention, 3 patients with incomplete clinical data, and 61 patients without blood samples collection. Finally, 124 patients were recruited into the current study, including 2 patients with ICH after 24 h, 4 patients with asymptomatic ICH in 24 h, 7 patients with sICH in 24 h, and 111 patients without any ICH in 24 h after IVT. There was no significant difference in the baseline characteristics between included patients and excluded patients (Table 1). With a ratio of 1:1, 7 patients without any ICH and 7 patients with sICH were matched to the non-sICH group and sICH group for final analysis (Figure 1). There was no significant difference in the baseline characteristics between the two groups (Table 2).

To the best of our knowledge, this is the first report that identified significant changes in 9 serum biomarkers in a sICH group compared with those in a non-sICH group, including increased biomarkers, such as BDNF, CCL-24, IL-6, IL-10, IL-18, and VEGF, and decreased biomarkers, such as CCL-11, ICAM-1, and IL-7.

There have been a lot of studies to investigate the association of biomarkers with post-thrombolytic hemorrhagic transformation in stroke (6–11). However, these studies have not investigated changes in serum biomarkers before and after sICH in thrombolytic stroke. Our results showed that these significantly changed biomarkers mainly contributed to the inflammatory response. As a result of recruited leukocytes and released proinflammatory cytokines, inflammation contributed to brain injury following ICH (16). Our results indicate that neuroinflammatory responses play an important role in post-thrombolytic sICH.

In the present study, we found significantly decreased circulating levels of IL-7, ICAM-1, and CCL-11 in the sICH patients, compared with non-sICH patients, which is consistent with previous studies (17–19). After stroke injury, peripheral leukocytes migrate into injured brain tissue through adhesion molecules and chemokines (20). As an adhesive protein, ICAM-1 facilitates leukocyte adhesion to vascular endothelium and entry into brain tissue through the blood-brain barrier (21–24). The CCL-11 is a proinflammatory cytokine and induces chemotaxis of leukocytes into the injured brain region (24). IL-7 mainly activates recruited T lymphocytes and further produces proinflammatory factors (25–27). As ICAM-1, CCL-11, and IL-7 were widely involved in transmigration and activation of leukocytes, we hypothesized that a decrease in the three biomarkers could be due to their significant consumption of biomarkers by migrating into injured brain tissue along with leucocytes after sICH. Additionally, tPA was reported to induce a surge of circulating immune cells and mobilize peripheral neutrophils and T cells (28), which may also be involved in the neuroinflammatory response.

Our study also found that CCL-24, IL-6, and IL-18 significantly increased after sICH, compared with patients with non-sICH. These biomarkers act as proinflammatory factors and express in the recruited leukocytes and activated glia. For example, CCL-24 was found to act directly on chemokine receptor CCR-3 to recruit leukocytes and play a role in mediating the neuroinflammation set off by ischemic-reperfusion injury (29, 30). IL-6 was produced in activated microglia and caused the brain injury after intracerebral hemorrhage under inflamed conditions (31, 32). IL-18, produced by activated macrophages and involved in neuroinflammation (33), was correlated with poor outcomes in intracerebral hemorrhage (34). However, as shown in Table 2, the concentration of IL-6 significantly increased after IVT in both the sICH and non-sICH groups, which was not consistent with the other two biomarkers. These results suggested that IL-6 may be triggered to secrete by ischemic or hemorrhagic brain injury, while CCL-24 and IL-18 were specifically related to sICH.

In addition, BDNF, IL-10, and VEGF were significantly increased after sICH, which was consistent with previous studies (35–37). BDNF, secreted from microglia, was found to be involved in the endogenous neuroprotection after brain injury (35, 38–40). As an anti-inflammatory cytokine, IL-10 is secreted mainly by the lymphocytes and monocytes and it inhibits transcription and production of the proinflammatory factor IL-6 (41, 42), which was reported to be associated with better neurological prognosis (43). Additionally, VEGF was an important angiogenesis regulatory factor and mainly enhanced vascular permeability and promoted endothelial cell proliferation after ICH (44). As shown in Table 2, the concentrations of IL-10 and VEGF increased in two groups, which was not consistent with BDNF. These results suggest that as a self-defending mechanism, the secretion of IL-10 and VEGF may be over-released from the glia and leukocytes in response to ischemic or hemorrhagic brain injury, while BDNF may be specifically triggered by hemorrhagic insult.

Unexpectedly, we did not detect significant changes in other preset biomarkers, such as MMP-9 and ferritin, which were previously reported to be associated with post-thrombolytic hemorrhagic transformation in ischemic stroke (6). The negative results may be attributed to the following reason. Most previous studies mainly detected the levels of biomarkers at admission (45, 46), while the present study focused on investigating the change from admission to 24 h later.

Based on functional enrichment analysis, cell adhesion positive regulation, cytokine activity, extracellular matrix, and cytokine–cytokine receptor interaction were significantly enriched items. After a stroke, adhesion molecules (e.g., ICAM-1) mediated several leukocytes and cytokines (e.g., VEGF and IL-7) entered into the brain through the injured blood-brain barrier (20, 22). The chemokines (e.g., CCL-11 and CCL-24) recruited leukocytes to mediate activation and release of proinflammatory cytokines (e.g., IL-6 and IL-18) (47). In addition, the cytokine–cytokine receptor interaction between CCL-24 and CCR-3 mediated the recruitment of leukocytes (29). Therefore, we inferred that cell adhesion regulation, cytokine activity, and cytokine–cytokine receptor interaction may contribute to brain injury after ICH through mediating recruitment of leukocytes and releasing of proinflammatory cytokines.

We acknowledge that our study has several limitations. First, it is a small-sample study with only 7 AIS patients with post-thrombolytic sICH in 24 h and collecting blood samples. Additionally, the study only collected blood samples before and 24 h after IVT. A large cohort study with samples collected at more time points (e.g., 2, 6, and 12 h after IVT) is warranted. Second, only serum biomarkers were measured in the present study. Given the neuroinflammation-related or neuroprotection-related biomarkers, it may provide valuable information if these biomarkers are measured in the brain such as CSF. Third, the sensitivity and specificity of identified biomarkers for predicting sICH need to be validated in the future. Fourth, the change of biomarkers in patients with different etiologies should be investigated in the future. Finally, although baseline data were matched through propensity scores matching, some potential biases may be inevitable. For example, there may exist an imbalance in the baseline circulating levels of biomarkers.

For the first time, the present study prospectively investigated the changes of preset 49 serum biomarkers in patients with ischemic stroke receiving thrombolysis and found that 6 neuroinflammatory and 3 neuroprotective biomarkers were involved in the brain injury following post-thrombolytic sICH. The role of these biomarkers warrants further investigation given the potential therapeutic implications.

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 Institution Human Research Ethics Committees of General Hospital of Northern Theater Command. The patients/participants provided their written informed consent to participate in this study.

H-SC designed and reviewed. YC conducted the analyses and drafted the manuscript. X-HW, YZ, S-YC, B-YS, and L-HW contributed to the implementation of blood sample collection. All authors contributed to the article and approved the submitted version.

The study was funded by grants from the National Key R&D Program of China (2017YFC1308203) and the Project on Research and Application of Effective Intervention Techniques for Chinese Stroke Guidelines from the National Health and Family Planning Commission in China (GN-2016R0008).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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