Fig 1: Regional MMP-9 plasma concentrations, neutrophil MMP-9 expression, and MMP activity with plasma concentrations stratified by type of intracranial bleeding and functional outcome category. Semi-log plots of MMP-9 plasma concentrations (a, d, and e), fluorescence intensity of neutrophils stained for MMP-9 (b; presented with examples at single-cell level, scale bar in white = 10 μm), and gelatine zymography experiments (c) in arterial non-occlusive control samples at cervical level of the internal carotid artery (systemic) vs samples from centrally within the cerebral collateral circulation (ischaemic) in human large-vessel ischaemic stroke. Zymography bands (c) were obtained from n = 10 pooled plasma samples. Zymographic intensity of 86 kDa MMP-9 and 72 kDa MMP-2, respectively. Intracranial bleedings (d) were anatomically categorized according to the Heidelberg Bleeding Classification (HBC: 1a, haemorrhagic infarction (HI1)—scattered small petechiae; 1b, HI2—confluent petechiae; 1c, parenchymal haematoma (PH1)—haematoma within infarcted tissue, occupying <30%; 2, PH2—haematoma occupying ≥30% of the infarcted tissue; 3a—PH remote from infarcted brain tissue; 3b—intraventricular haemorrhage; 3c—subarachnoid haemorrhage; 3d—subdural haemorrhage) and grouped with respect to the type of bleeding (HBC classes 1a + b, minor [petechial] intracerebral haemorrhage; HBC classes 1c–3a, major intracerebral haemorrhage [parenchymal haematoma]; HBC classes 3b–d, intracranial-extracerebral haemorrhage). Functional outcome (e) was assessed by the modified Rankin Scale (mRS: mRS≥5, severe disability or death at hospital discharge). The long horizontal bar shows the median value, and the short error bar shows the interquartile range. Data were analysed using Wilcoxon matched-pairs signed rank test (a, b, d, and e), Kruskal–Wallis test with Benjamini, Krieger and Yekutieli post hoc analysis (d), and Mann–Whitney test (e). Individual p values are given for p < 0.05. CTCF, corrected total cellular fluorescence; HBC, Heidelberg Bleeding Classification; kDa, kilo Dalton; MMP-9/-2, Matrix Metalloproteinase-9/2; mRS, modified Rankin Scale; ng/ml, nanograms per millilitre; ns, not significant.
Fig 2: ROC analysis of MMP-9 plasma concentrations for discrimination between patients at low and high risk of major intracerebral haemorrhages, and severe disability or death. ROC curves of regional ischaemic (blue) and systemic (red) MMP-9 plasma concentrations in prediction of HBC1c-3a bleedings (a), and mRS ≥5 at hospital discharge (b) in large-vessel ischaemic stroke; categorisation corresponding to Fig. 2. Green dots represent values of maximised balances between sensitivity and specificity. Reported p values for the differences in AUCs were derived from DeLong's test. Youden index, HBC1c–3a: MMP-9ischaemic 552.9 ng/ml, 72.2% sensitivity (95% CI 46.5–90.3%), 66.7% specificity (95% CI 57.2–75.2%); MMP-9systemic 448.9 ng/ml, 55.6% sensitivity (95% CI 30.8–78.5%), 68.4% specificity (95% CI 59.1–76.8%); Youden index, mRS ≥5: MMP-9ischaemic 363.3 ng/ml, 75.9% sensitivity (95% CI 62.8–86.1%), 54.1% specificity (95% CI 42.1–65.7%); MMP-9systemic 235.5 ng/ml, 70.7% sensitivity (95% CI 57.3–81.9), 51.4% specificity (95% CI 39.4–63.1). AUC, area under curve; HBC, Heidelberg Bleeding Classification; MMP-9, Matrix Metalloproteinase-9; ng/ml, nanograms per millilitre; mRS, modified Rankin Scale; ROC, receiver operating characteristic.
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