Fingolimod reduces hemorrhagic transformation associated with delayed tissue plasminogen activator treatment in a mouse thromboembolic model

Abstract

Background and purpose: The sphingosine 1-phosphate receptor agonist fingolimod reduces infarct size in rodent models of stroke and enhances blood-brain barrier integrity. Based on these observations, we hypothesized that combination of fingolimod with tissue plasminogen activator (tPA) would reduce the risk of hemorrhagic transformation associated with delayed administration of tPA.

Methods: We evaluated the effects of fingolimod in a mouse model of thromboembolic stroke, in which both the beneficial effect of reperfusion associated with early tPA treatment and hemorrhagic transformation associated with delayed administration mimic clinical observations in humans.

Results: Our results demonstrate that fingolimod treatment attenuates the neurological deficit and reduces infarct volume after in situ thromboembolic occlusion of the middle cerebral artery. Combination of fingolimod and tPA improves the neurological outcome of the thrombolytic therapy and reduces the risk of hemorrhagic transformation associated with delayed administration of tPA.

Conclusions: This study confirms the protective efficacy of fingolimod as a treatment against ischemic stroke in another rodent model of stroke (thromboembolic occlusion), and suggests that fingolimod could potentially be used in combination with tPA to reduce the risk of brain hemorrhage.

Figure 1

A) Analysis of cerebral blood flow by laser speckle flowmetry (LSF) in the sham group (18% glycerol injection), in animals receiving thrombin injection (ischemia) and animals with early tPA-induced recanalization (ischemia + tPA). Cerebral blood flow is expressed as percent of basal level. B) Regional cerebral blood flood using [¹⁴C]iodoantipyrine autoradiography was performed 45 min after 18% glycerol injection (sham, n=2); 45 min (n=2) or 240 min (n=4) after MCA occlusion (note that the ischemic area extends beyond the territory of the occluded MCA branch, probably due to the occurrence of CSD/peri-infarct depolarization); 4 hrs after tPA injection in mice undergoing early (30 min, n=2) or late (180 min, n=2) tPA recanalization.

Figure 2

A) Representative H&E-stained sections illustrating the effect of vehicle or fingolimod (0.5 mg/kg FTY720) on infarct volume in animals with permanent occlusion, early recanalization (tPA 30 min after occlusion) and delayed recanalization (tPA 3 hours after occlusion). B) Infarct volumes were measured 3 days after occlusion. Data are means ± SD; n=9 to 10; *P<0.05.

Figure 3

Effect of vehicle and fingolimod (FTY720, 0.5 mg/kg) on functional deficit, evaluated by the grid walking test in the three experimental groups tested. Panels A and B show the percentage of footslips for the left and right forepaws. The grid walking test was performed 3 days after occlusion. C shows the effect of vehicle and fingolimod (FTY720, 0.5 mg/kg) on functional deficit, evaluated by the cylinder test in the three experimental groups tested. This test was performed before surgery (baseline) and 3 days after occlusion. Animals treated with FTY720 show a behavior close to baseline. Data are means ± SD; n=9 to 10; *P<0.05.

Figure 4

A) Representative DAB-stained sections illustrating the effect of vehicle or fingolimod (0.5mg/kg FTY720) on hemorrhage volume in animals with permanent occlusion, and animals with early or delayed recanalization. B) The volume of hemorrhages (small arrows in A) was analyzed 3 days after occlusion. Panel C represents the ischemic region normalized for infarct volumes. Data are means ± SD; n=9 to 10; *P<0.05.

Figure 5

A) Effect of fingolimod (0.5 mg/kg FTY720) on Evans Blue extravasation in animals with delayed recanalization determined in contralateral and ipsilateral hemisphere of control and treated group. B shows Evans Blue extravasation in ischemic region normalized for infarct volume. Evans Blue extravasation was determined 24 h after occlusion. Data are means ± SD; n=6; *P<0.05.