Intravenous thrombolysis with recombinant tissue plasminogen activator (tPA) is the mainstay therapeutic method of acute ischemic stroke (AIS) with proven clinical benefit (1) and is recommended up to 4.5 h after stroke onset (2). In addition, recent randomized controlled trials and meta-analyses enabled the extension of the time window in selected cases or administration of rtPA in some subjects with unknown onset of stroke (3–5).

The most common complication of rtPA treatment is the hemorrhagic transformation of the ischemic lesion. This transformation might be of various severity that has been classified with ECASS-II score (Table 1) (6). The severity of hemorrhagic transformation is strictly connected with clinical significance and the symptomatic intracerebral hemorrhage (sICH), which occurs in 3–7% of rtPA treated patients, is of the highest clinical interest since it is associated with poor functional outcomes. Symptomatic intracerebral hemorrhage has been well-characterized in the multiple studies (2, 7–9).

Administration of rtPA might also result in another hemorrhagic complication—cerebral microhemorrhages or microbleeds (CMBs). CMBs are small, rounded signal loss lesions surrounded by brain tissue with a diameter up to 5 mm. CMBs might appear remotely from the ischemic lesion in an isolated or diffuse pattern across the brain. The CMBs neuroimaging characteristics are listed in Table 2 (10–13). The prevalence of CMBs is significantly higher in the elderly population with multiple comorbidities and high-total cardiovascular risk, especially hypertension with subsequent hypertensive arteriopathy (13, 14). Usually, CMBs are clinically silent in the terms of acute stroke care but might have a cumulative impact on patients in the following years, mainly because of the associations between the load of CMBs and cognitive impairment (15–20).

Cerebral microbleeds have also been under study because of their possible pathophysiological connection with hemorrhagic transformation as CMBs are commonly treated as markers of increased vascular vulnerability due to severe small vessel disease (21–23). This connection seems plausible also in light of the recent analyses that have revealed that CMBs burden might be a useful single marker of the risk of sICH in patients, after stroke or TIA, receiving oral anticoagulants (OACs) (24, 25). In these reports, CMBs burden was more predictive of sICH than other tools, including HAS-BLED (26).

The problem of rtPA-associated CMBs has already been addressed in a few studies (27–32). However, only four of them were planned to assess true baseline CMBs load with MRI preceding the rtPA administration (28–31). The problem to quantify and compare CMBs before and after the thrombolytic treatment is related to the fact that their proper assessment requires the use of MRI. The long duration of standard MRI testing interferes with the urge of administering the rtPA and thus precludes regular and easy use of this neuroimaging technique in thrombolysed patients.

In the presented study, we investigated whether administration of rtPA in AIS results in the appearance of new CMBs and whether the baseline CMBs load increases the risk of new CMBs and hemorrhagic transformation after the rtPA treatment. The assessment was performed using pre- and post-thrombolysis special MRI protocols.

On initial T2^*-EPI-GRE we identified 37 baselines CMBs in 14 patients (28.6%). A total of thirty-four (91.9%) of CMBs were localized in the cortical/subcortical area and only 3 (8.1%) in the deep brain structures. In all but one patient, the number of baseline CMBs did not exceed 3. Interobserver agreement was high and kappa values are presented in Table 5. On follow-up T2^*-EPI-GRE, the total amount of CMBs increased up to an absolute number of 103 (all patients combined). New CMBs were found in 5 (14.3%) of 35 patients without baseline CMBs (Figure 1) and in 9 (64.3%) of 14 with CMBs detected in baseline MRI. Among a total of 66 new CMBs, 48 (72.7%) were localized in the cortical/subcortical area, 18 (27.3%) in deep brain structures, and 35 (53.0%) in the ipsilateral hemisphere. Hemorrhagic transformation (ECASS) was observed in 10 (20.4%) patients - 6 HI1, 2 HI2, 1 PH1, and 1 PH2. Baseline CMBs did not correlate significantly with hemorrhagic transformation (p = 0.647).

Patients with new CMBs were older (median 72.5, interquartile range (IQR) 66.5, 84.0 vs. 62.0, IQR 46.0, 75.5; p = 0.026), had higher creatinine level (median 1.0, IQR 0.9,1.1 vs. 0.8, IQR 0.7, 0.9; p = 0.035) and more often had higher counts of baseline CMBs: 9 (64.3%) vs. 5 (14.3%), p < 0.001. Other parameters were not statistically significantly different between the groups including pre-existing hypertension, the National Institutes of Health Stroke Scale (NIHSS) admission score, stroke subtype according to TOAST classification, the severity of leukoaraiosis, and baseline DWI lesion volume.

Multiple logistic regression analysis (Table 6) indicated that presence of baseline CMBs (risk ratio 5.95, 95% confidence interval CI 2.69–13.20, p < 0.001) and lower platelets level (risk ratio 0.992, 95% CI 0.986–0.998, p = 0.007) were independently associated with new CMBs.

In our patient sample, administration of rtPA resulted in the appearance of new CMBs in nearly 30% of the subjects with AIS, and the baseline CMBs were associated with the higher risk of the new ones appearing after the treatment. However, in this cohort, the baseline CMB load was not related to the increased risk of hemorrhagic transformation, unlike in many of the previous reports (21, 23, 32, 39). This finding seems not to be in line with the pathophysiological connections of CMBs and various vascular pathologies, notably small vessel disease, which in turn is related to increased vascular vulnerability and thus with hemorrhagic transformation—these correlations have been extensively discussed elsewhere (21–23). The reason for this discrepancy might be that these associations may be influenced by many confounding factors connected with the individual characteristics of a patient. CMBs are to be found in “healthy” populations, however, the insight in the available reports prove susceptibility of these populations for numerous future health risks that are additionally modulated by many coexisting factors such as hypertension, smoking, Apo E homozygosity, aspirin intake, white matter lesions or cerebral amyloid angiopathy (40). In addition, it has been reported that the correlation between CMBs and hemorrhagic transformation is not linear and that there is a threshold of baseline CMBs that, only when exceeded, results in higher risks for the transformation, especially for the sICH. The most commonly reported threshold is the > 10 CMBs (41) and the definite majority of our patients presented with not more than 3 baseline CMBs.

One of the future health issues of patients with CMBs is cognitive decline. The available studies suggest strong correlations between CMBs and cognitive impairment (15, 16). The substantial correlation between rtPA treatment and new CMBs found in our study, may have a major implication for the cognitive health of patients with AIS, and thus, longitudinal long-term studies in rt-PA treated patients with AIS are warranted for the need of powerful assessment of potential relations between thrombolytic treatment, CMBs, and cognitive function. This seems to be specifically important in patients with minor and/or lacunar stroke, where thrombolytic treatment does not have that much scientific data, as large vessel occlusion strokes, to prove favorable outcomes (42).

The study was not controlled with a placebo sample and therefore the direct correlation of new CMBs with rtPA treatment cannot be concluded. Additionally, the acute cerebral infarction itself is suggested to promote the development of CMBs (43). However, taking into account other studies on CMBs in acute stroke, and the known rate of the hemorrhagic infarction unrelated to rtPA administration, it is unlikely that this burden value would be as high as about 30% with no connection to rtPA. Furthermore, a strong indication of rtPA-CMBs correlation has been presented in the recent study by Miwa et al., where new CMBs were found only in patients who received rtPA (31). The high percentage of new CMBs in our study is much higher in comparison with other studies that revealed rates in the range of 4–13% (27–31). We believe that it is probably connected with a small sample size that resulted in incidental recruitment of more predisposed patients, as we discussed earlier.

This study has several methodological limitations. Importantly, as discussed earlier, we do not have a control group of non-thrombolysis patients to compare with. Another limitation of this study was the use of a 1.5-T field MR machine which is inferior to a 3-T in detecting CMBs, and the T2^*EPI-GRE imaging sequence—much shorter but less sensitive than SWI for CMBs detection (44, 45). To support the lower sensitivity of T2^*EPI-GRE sequences, SWI sequences were additionally used to confirm the CMB assessments. Finally, our study encompassed a relatively small sample of subjects, which is, however, similar to many of the reported cohorts. The main reason for the small number of patients is the conflict of interest between the optimal timing of rtPA treatment and the demanding circumstances of MRI testing that is necessary for proper CMBs assessment.

In conclusion, in this study, we present that baseline CMBs burden in patients with AIS is associated with a higher risk of new CMBs after rtPA treatment, but not with the risk of hemorrhagic transformation. New CMBs alone or combined with other selected characteristics (e.g., age, platelet count, or creatine level as indicated in this study) may become a useful predictor of long-term CMBs-related complications of thrombolytic treatment of stroke.

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Independent Bioethics Committee for Scientific Research at Medical University of Gdańsk, Poland (Approval No. NKBBN/76/19). The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.

BJ co-conceptualization of the study, patient recruitment, data assessment and analysis, combined analysis of all data, and writing. AG-G patient recruitment, data assessment and analysis, writing, and editing. DO neuroimaging data collection and assessment and writing. ES neuroimaging data assessment. AW statistical analysis and writing. BR clinical data assessment. BK conceptualization and funding receipt (a major project), writing, review, editing, and coordination. All authors contributed to the article and approved the submitted version.

Neuroimaging data were part of our project funded by Siemens Heathineers (research grant for the Medical University of Gdansk, PI – BK, 2018; Contract Number C00229230). The authors declare that this study received funding from Siemens Heathineers. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

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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