Pro-inflammatory mediators and apoptosis correlate to rt-PA response in a novel mouse model of thromboembolic stroke

Abstract

Background: A recent study suggests that patients with persistent occlusion of the middle cerebral artery (MCA) following treatment with recombinant tissue plasminogen activator (rt-PA) have better outcomes than patients with MCA occlusion not receiving rt-PA. We performed a study to elucidate possible mechanisms of this finding in a new model of thromboembolic stroke closely mimicking human pathophysiology.

Methods: Thromboembolic stroke was induced by local injection of thrombin directly into the right MCA of C57 black/6J mice. Rt-PA was administered 20 and 40 min after clot formation. The efficiency of rt-PA to induce thrombolysis was measured by laser Doppler. After 24 h, all animals were euthanized and interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), matrix metalloproteinase (MMP)-9, Caspase-3, hsp 32 and hsp 70 protein levels were investigated by immunofluorescence. Presence of hemorrhage was verified and infarct volume was measured using histology.

Results: Thrombin injection resulted in clot formation giving rise to cortical brain infarction. Early rt-PA treatment starting at 20 min after the clot formation resulted in 100% recanalization. However, rt-PA-induced thrombolysis dissolved the clot in only 38% of the animals when administered 40 min after clot formation. Protein levels of IL-6, TNF-α, MMP-9, Caspase-3, hsp 32 and hsp 70 were increased after MCAO, whereas treatment with rt-PA attenuated the expressions of inflammatory markers in those animals where the thrombolysis was successful. In addition, the infarct size was significantly reduced with rt-PA treatment compared to non-treated MCAO, regardless of whether MCA thrombolysis was successful.

Conclusions: The present study demonstrates a clear correlation of the protein expression of inflammatory mediators, apoptosis and stress genes with the recanalization data after rt-PA treatment. In this model rt-PA treatment decreases the infarct size regardless of whether vessel recanalization is successful.

Figure 1. High resolution MR angiography of mouse brain before (a) and after (b) right MCA occlusion (arrows).

T2-weighted images (c) and ADC-maps (d) demonstrating infarction.

Figure 2. Effect of treatment with rt-PA on CBF after MCAO in mice.

(a), MCAO+rt-PA after 20 min and 40 min, (b), MCAO+rt-PA after 40 min. Data were obtained by laser Doppler flowmetry and are expressed as mean ± s.e.m. values, n = 5–9 *P≤0.05, **P≤0.01, ***P<0.001 significant difference between treated groups and MCAO groups. Statistical analyses were performed using Kruskal-Wallis non-parametric test together with Dunn’s post-hoc test.

Figure 3. Size of infarct volume.

Treatment with rt-PA significantly reduced the infarct volume compared to MCAO. Data are expressed as mean± s.e.m.; n = 5–9 *P≤0.05, significant difference between treated groups and MCAO groups. Statistical analyses were performed using Kruskal-Wallis non-parametric test together with Dunn’s post-hoc test.

Figure 4. Effect of administration of rt-PA on protein expression.

Sections from the infarction core showing IL-6, TNF-α, Caspase 3, MMP-9, and hsp 70 protein expressions. The images represent the control group (A, D, G, J, M and P), MCAO (B, E, H, K, N, Q) and MCAO+rt-PA treatment (C, F, I, L, O, R). There are significant increases in IL-6, TNF-α, Caspase 3 and hsp 70 protein levels in the MCAO group compared to the control group. Treatment with rt-PA prevented the increase in expression of these proteins in those animals that recanalized after treatment. There was a slight increase in hsp 32 and MMP-9 protein expression in ischemic brain tissue as compared to control. Data were obtained with confocal microscopy.

Figure 5. Bar graphs showing semi-quantification of fluorescence intensity for IL-6, TNF-α, Caspase 3, MMP-9, hsp 70 and hsp 32 proteins.

Data are presented as the mean percentage relative to control ± s.e.m.; n 5 = 9 *P≤0.05, significant difference between control groups and MCAO, # P≤0.05, significant difference between treated groups and MCAO groups. Statistical analyses were performed using Kruskal-Wallis non-parametric test together with Dunn’s post-hoc test.