Atorvastatin extends the therapeutic window for tPA to 6 h after the onset of embolic stroke in rats

Abstract

We investigated the neuroprotective effect of atorvastatin in combination with delayed thrombolytic therapy in a rat model of embolic stroke. Rats subjected to embolic middle cerebral artery (MCA) occlusion were treated with atorvastatin at 4 h, followed by tissue plasminogen activator (tPA) at 6 or 8 h after stroke. The combination of atorvastatin at 4 h and tPA at 6 h significantly decreased the size of the embolus at the origin of the MCA, improved microvascular patency, and reduced infarct volume, but did not increase the incidence of hemorrhagic transformation compared with vehicle-treated control animals. However, monotherapy with tPA at 6 h increased the incidence of hemorrhagic transformation and failed to reduce infarct volume compared with the control group. In addition, adjuvant treatment with atorvastatin at 4 h and with tPA at 6 h reduced tPA-induced upregulation of protease-activated receptor-1, intercellular adhesion molecule-1, and matrix metalloproteinase-9, and concomitantly reduced cerebral microvascular platelet, neutrophil, and fibrin deposition compared with rats treated with tPA alone at 6 h. In conclusion, a combination of atorvastatin and tPA extended the therapeutic window for stroke to 6 h without increasing the incidence of hemorrhagic transformation. Atorvastatin blocked delayed tPA-potentiated adverse cerebral vascular events, which likely contributes to the neuroprotective effect of the combination therapy.

Figure 1

Infarct volume. Bar graph shows the effects of monotherapy with tPA and atorvastatin and the combination therapy on infarct volume assessed 7 days after MCA occlusion. Values are mean ± s.e. *P < 0.05 as compared with that of the saline-treated group. ^†P < 0.05 as compared with that of the tPA alone group. ^‡P < 0.05 as compared with that of the atorvastatin alone group.

Figure 2

Embolus at the origin of the occluded MCA. (A) Schematic representation of a ventral view of a rat brain is shown. The enlarged boxed area from panel A shows the intracranial segment of the ICA and the MCA. A large fragment of Evans blue dye (B, red) was detected within the origin of the MCA and intracranial segment of the ICA, which blocked plasma perfusion (panel B, green) from a representative rat treated with tPA alone. The combination treatment with atorvastatin at 4 h and tPA at 6 h resulted in only a small fragment of Evans blue dye (C, red) at the origin of the MCA and the intracranial segment of the ICA, which were perfused by FITC-dextran (panel C, green). (D) shows the quantitative data of an embolus (n=4 per group). Bar in panels B and C=400 μm.

Figure 3

Thrombocyte, fibrin/fibrinogen, MPO, ICAM-1, MMP9, and collagen type IV immunoreactive cerebral vessels and FITC-dextran perfusion. Double immunofluorescent staining (A–F) shows thrombocyte (panels A and B, green), fibrin/fibrinogen (panels C and D, green), and MPO (panels E and F, green) in cerebral blood vessels (Eba, panels A–F, red) of representative rats treated with tPA alone (panels A, C, and E) and with a combination of atorvastatin at 4 h and tPA at 6 h (panels B, D, and F). (G–L) The cerebral microvessels perfused with FITC-dextran in a normal rat (panel H), and in representative rats treated with saline (panel I), tPA (panel J), atorvastatin (panel K), and a combination of tPA at 4 h and atorvastatin at 6 h (panel L) 30 h after MCA occlusion (n=4 per group) is shown. (M–P) Represents the quantitative data of the number of thrombocyte (panel M), fibrin/fibrinogen (panel N), MPO immunoreactive cerebral vessels (panel O), and microvessels perfused with FITC-dextran (panel P). Double immunostaining shows that cerebral blood vessels with diffused collagen type IV immunoreactivity (Q, R, T, U; red) were ICAM-1 (panels Q and R, green) or MMP9 positive (panels T and U, green) in representative rats treated with tPA alone (panels Q and T), and with a combination of atorvastatin at 4 h and tPA at 6 h (panels R and U). Panel S and (V) show intensive collagen type IV immunoreactivity (red) in contralateral homologous areas wherein ICAM-1 (panel C, green) and MMP9 (panel F, green) were absent from a representative rat treated with tPA alone. Double immunostaining (W–Y) shows that ICAM-1 (panels W and Y, green; arrows) and MMP9 (panels X and Y, brown; arrowheads) immunoreactivities were in different vessels of a representative rat treated with tPA alone. Panel Y is a merged image. Ctx, cortex; Str, striatum; CC, corpus callosum. *P < 0.05 as compared with that of saline-treated group. ^†P<0.05 as compared with that of the tPA alone group. ^‡P<0.05 as compared with that of the atorvastatin alone group. ^§P<0.05 for treatment interaction (synergistic effect). Bars for panels A–F=40 μm; panels H–L=1 mm; panels Q–Y=40 μm.

Figure 3

Thrombocyte, fibrin/fibrinogen, MPO, ICAM-1, MMP9, and collagen type IV immunoreactive cerebral vessels and FITC-dextran perfusion. Double immunofluorescent staining (A–F) shows thrombocyte (panels A and B, green), fibrin/fibrinogen (panels C and D, green), and MPO (panels E and F, green) in cerebral blood vessels (Eba, panels A–F, red) of representative rats treated with tPA alone (panels A, C, and E) and with a combination of atorvastatin at 4 h and tPA at 6 h (panels B, D, and F). (G–L) The cerebral microvessels perfused with FITC-dextran in a normal rat (panel H), and in representative rats treated with saline (panel I), tPA (panel J), atorvastatin (panel K), and a combination of tPA at 4 h and atorvastatin at 6 h (panel L) 30 h after MCA occlusion (n=4 per group) is shown. (M–P) Represents the quantitative data of the number of thrombocyte (panel M), fibrin/fibrinogen (panel N), MPO immunoreactive cerebral vessels (panel O), and microvessels perfused with FITC-dextran (panel P). Double immunostaining shows that cerebral blood vessels with diffused collagen type IV immunoreactivity (Q, R, T, U; red) were ICAM-1 (panels Q and R, green) or MMP9 positive (panels T and U, green) in representative rats treated with tPA alone (panels Q and T), and with a combination of atorvastatin at 4 h and tPA at 6 h (panels R and U). Panel S and (V) show intensive collagen type IV immunoreactivity (red) in contralateral homologous areas wherein ICAM-1 (panel C, green) and MMP9 (panel F, green) were absent from a representative rat treated with tPA alone. Double immunostaining (W–Y) shows that ICAM-1 (panels W and Y, green; arrows) and MMP9 (panels X and Y, brown; arrowheads) immunoreactivities were in different vessels of a representative rat treated with tPA alone. Panel Y is a merged image. Ctx, cortex; Str, striatum; CC, corpus callosum. *P < 0.05 as compared with that of saline-treated group. ^†P<0.05 as compared with that of the tPA alone group. ^‡P<0.05 as compared with that of the atorvastatin alone group. ^§P<0.05 for treatment interaction (synergistic effect). Bars for panels A–F=40 μm; panels H–L=1 mm; panels Q–Y=40 μm.