Early Abrogation of Gelatinase Activity Extends the Time Window for tPA Thrombolysis after Embolic Focal Cerebral Ischemia in Mice

Abstract

Acute ischemic stroke (AIS) is caused by clotting in the cerebral arteries, leading to brain oxygen deprivation and cerebral infarction. Recombinant human tissue plasminogen activator (tPA) is currently the only Food and Drug Administration-approved drug for ischemic stroke. However, tPA has to be administered within 4.5 h from the disease onset and delayed treatment of tPA can increase the risk of neurovascular impairment, including neuronal cell death, blood-brain barrier (BBB) disruption, and hemorrhagic transformation. A key contributing factor for tPA-induced neurovascular impairment is activation of matrix metalloproteinase-9 (MMP-9). We used a clinically-relevant mouse embolic model of focal-cerebral ischemia by insertion of a single embolus of blood clot to block the right middle cerebral artery. We showed that administration of the potent and highly selective gelatinase inhibitor SB-3CT extends the time window for administration of tPA, attenuating infarct volume, mitigating BBB disruption, and antagonizing the increase in cerebral hemorrhage induced by tPA treatment. We demonstrated that SB-3CT attenuates tPA-induced expression of vascular MMP-9, prevents gelatinase-mediated cleavage of extracellular laminin, rescues endothelial cells, and reduces caveolae-mediated transcytosis of endothelial cells. These results suggest that abrogation of MMP-9 activity mitigates the detrimental effects of tPA treatment, thus the combination treatment holds great promise for extending the therapeutic window for tPA thrombolysis, which opens the opportunity for clinical recourse to a greater number of patients.

Keywords: MMP-9; SB-3CT; cerebral ischemia; endothelial transcytosis; matrix; tissue plasminogen activator.

Figure 1.

Effect of SB-3CT on brain damage in tPA-treated mice after embolic ischemia. A, Experimental design for embolic MCAO and SB-3CT and tPA administration. SB-3CT was given intraperitoneally at 25 mg/kg; tPA was administered intravenously at 2.5 mg/kg. Vehicle controls (25% DMSO/65% PEG-200/10% water intraperitoneally and saline intravenously) were included. B, Representative cresyl violet-stained images at different distances from bregma showing infarct areas (light blue regions marked by black lines). Infarct percentage for tPA given at 4 h (C) or 6 h (D) postischemia; n = 10 mice for vehicle, n = 6 for tPA, and n = 7 for SB-3CT + tPA groups; *p < 0.05 by one-tailed Student’s t test. Data are expressed as mean ± SEM.

Figure 2.

Effect of SB-3CT on BBB leakage in tPA-treated mice after embolic ischemia. A, BBB leakage as ascertained by IgG extravasation. Representative brain sections from the same mice described in Figure 1 were stained with goat anti-mouse IgG. IgG extravasation for tPA given at 4 h (B) or 6 h (C) postischemia; *p < 0.05 by one-tailed Student’s t test.

Figure 3.

Effect of SB-3CT on ICH in tPA-treated mice after embolic ischemia. A, Representative brain sections from the same mice described in Figure 1 revealed ICH of different sizes in the ischemic areas after embolic MCAO in mice. Scale bar: 1 mm. B, C, Quantification of hemorrhagic volumes. ICH was evaluated in cresyl violet-stained brain sections by bright-field microscopy in mice treated with saline or tPA at 4 h (B) or 6 h (C) postischemia. ICH volume was quantified using the stereology technique, with systematic sampling of 25–30 serial sections per brain. Each section was separated by 200-µm interval along the anteroposterial axis of the mouse brain; n = 10 mice in the vehicle-treated, n = 6 mice in the tPA-treated, and n = 7 mice in the SB-3CT + tPA-treated groups. D, E, Hemorrhage number of different areas in mice treated with tPA at 4 h (D) or 6 h (E) postischemia.

Figure 4.

Effect of SB-3CT on tPA-induced MMP-9 expression in endothelial cells and vascular laminin after embolic ischemia. A, Colocalization of MMP-9-positive immunoreactivity (green) with endothelial marker CD31 (red) in the ischemic penumbra. 3D deconvolution was used to enhance sharpness and contrast of fluorescent images. White horizontal and vertical lines indicate the sections of interest in the 3D structures of vessels. Radial and tangential planes, as well as the cross sections of the representative images, were achieved by orthogonal sectioning analysis and shown at the bottom right of the images: the tangential sections (A₁), the radial sections (A₂), and the cross sections (A₃). B, Photomicrographs of the ischemic penumbra and corresponding regions in the contralateral hemisphere for the different treatment groups. Scale bars: 20 μm (A), 100 μm (B), and 50 μm (inset).

Figure 5.

Effect of SB-3CT on tPA-induced degeneration of vascular laminin and endothelial cells after embolic ischemia. A, Representative brain sections from the same mice described in Figure 4 were immunostained with anti-laminin antibody (green), endothelial cells with CD31 (red) and cell nuclei with Hoechst dye (blue). Scale bar: 100 μm. B, Density of laminin-positive vessels. C,Density of CD31-positive vessels; n = 3 for vehicle, n = 6 for tPA, and n = 6 for SB-3CT + tPA groups; *p < 0.05 by one-tailed Student’s t test. Data are expressed as mean ± SEM.

Figure 6.

Effect of SB-3CT on tPA-induced increase of caveolae-mediated transcytosis after embolic ischemia. A, Representative brain sections from the same mice described in Figure 4 were immunostained with caveolin-1 (green), CD31 (red), and Hoechst (blue) in the ischemic penumbra. 3D deconvolution was used to enhance the sharpness and contrast of fluorescent images. B, Immunofluorescent staining showed increased caveolae-1 expression in the ischemic penumbra. Caveolin-1 expression became more pronounced with tPA treatment and decreased with SB-3CT + tPA treatment. C, Magnified images from the different treatment groups. White arrows indicate areas of less caveolin-1 expression; yellow arrows indicate areas of enhanced caveolin-1 expression in the endothelial cells. Scale bars: 20 μm (A), 100 μm (B), and 25 μm (C).

Figure 7.

Model for MMP-9-mediated impairment of the neurovascular unit after ischemic stroke. A, Schematic diagram shows impairment of the neurovascular unit due to upregulation of MMP-9 activity after ischemic stroke and exogenous tPA administration. B, left, In the normal neurovascular unit, endothelial cells (light green) and pericytes (red) interact with a shared basal lamina (yellow). Tight junctions (dark green) seal the endothelial cells. Caveolae (blue) are expressed in low level. Right, In the impaired neurovascular unit after ischemic stroke, increased MMP-9 (purple) degrades the basal lamina, which in turn leads to the impairment of endothelial cells and deformation of pericytes. Detachment of the pericytes away from the endothelial cells contributes to the increase of caveolae. MMP-9 also breaks tight junctions. These mechanisms result in increased permeability of the BBB, followed by leakage of tPA to the abluminal side of the neurovascular unit and the brain parenchyma.