Intravenous tPA therapy does not worsen acute intracerebral hemorrhage in mice

Abstract

Tissue plasminogen activator (tPA) is the only FDA-approved treatment for reperfusing ischemic strokes. But widespread use of tPA is still limited by fears of inadvertently administering tPA in patients with intracerebral hemorrhage (ICH). Surprisingly, however, the assumption that tPA will worsen ICH has never been biologically tested. Here, we assessed the effects of tPA in two models of ICH. In a mouse model of collagenase-induced ICH, hemorrhage volumes and neurological deficits after 24 hrs were similar in saline controls and tPA-treated mice, whereas heparin-treated mice had 3-fold larger hematomas. In a model of laser-induced vessel rupture, tPA also did not worsen hemorrhage volumes, while heparin did. tPA is known to worsen neurovascular injury by amplifying matrix metalloproteinases during cerebral ischemia. In contrast, tPA did not upregulate matrix metalloproteinases in our mouse ICH models. In summary, our experimental data do not support the assumption that intravenous tPA has a deleterious effect in acute ICH. However, due to potential species differences and the inability of models to fully capture the dynamics of human ICH, caution is warranted when considering the implications of these findings for human therapy.

Figure 1. tPA activity measures.

(A) Human tPA but not saline activates mouse plasminogen. In this assay, tPA converts plasminogen to plasmin, then plasmin converts D-Val-Leu-Lys-7-Amino-4-Methylcoumarin to a fluorescent product, which was measured over 1 hr at 5 minute intervals using a fluorescence plate reader. Data are provided in arbitrary fluorescence units (A.U.). Data are mean±SEM, 3 measures per group and time point. (B) Thrombolytic activity of tPA was proven in-vivo in a standard model of thromboembolic middle cerebral artery occlusion in rats. tPA restored regional cerebral blood flow (rCBF) more rapidly than saline. Data are mean±SEM, 4 animals per group.

Figure 2. Collagenase-induced ICH volumes are not increased by administration of i.v. tPA.

(A) Brain sections showing hematoma at 30 min, 60 min and 24 hrs after ICH induction. ICH starts to occur within 30 min and has largely developed by 1 hr. (B) Hematoma volumes at 24 hrs after ICH induction (mean±SD). tPA did not alter hematoma sizes, but heparin significantly worsened ICH as compared to saline and tPA treatment. (C) Results of the reconfirmation study. No difference was observed between saline and tPA mice, but heparin significantly increased ICH volume (mean±SD) as compared to saline and tPA treatment.

Figure 3. Femtosecond laser-induced cortical microhemorrhage volumes are not increased by i.v. administration of tPA.

(A) Two-photon excited fluorescence in vivo image projections of fluorescently-labeled blood plasma spanning a 20 µm depth centered at the hemorrhage origin. Image stacks are shown before and after rupturing the wall of a single penetrating arteriole using tightly focused femtosecond laser pulses. Representative examples from the three different intravenous infusion groups (saline, tPA, heparin) are shown. Extravasated plasma is visualized by diffuse fluorescence and can be seen in the post hemorrhage images in a halo surrounding the target vessel. The dark core immediately adjacent to the target vessel is filled with red blood cells. Post-hemorrhage (B) red blood cell (RBC) and (C) blood plasma extravasation diameters (mean±SEM) are shown for the three treatments: saline (n = 9 microhemorrhages), tPA (n = 11), and heparin (n = 10). No difference was found between the saline and tPA group, but heparin significantly increased both red blood cell and blood plasma extravasation diameters.

Figure 4. Treatment with tPA worsened subarachnoid hemorrhage.

Blood volumes at 24 hrs after SAH induction (mean±SEM) are provided. tPA significantly increased SAH blood volume (tPA, n = 6; saline, n = 7).

Figure 5. Gelatin zymography of MMP responses.

(A) Representative zymogram gels showing MMP-2 and MMP-9 levels in brain homogenates derived from mice subjected to focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) or primary ICH mice. (B) Quantified densitometry of the zymogram results (arbitrary units normalized to constant loading of MMP standards). After 3 hrs of focal cerebral ischemia followed by 3 hrs reperfusion, tPA significantly amplified MMP-2 and MMP-9 levels. In contrast, there were no differences in MMPs in saline versus tPA-treated mice after ICH.