Fingolimod provides long-term protection in rodent models of cerebral ischemia

Abstract

Objective: The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species.

Methods: We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod.

Results: In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha.

Interpretation: These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Figure 1

Effects of FTY720 on cerebral blood flow. Regional cerebral blood flow was measured using a flexible laser Doppler probe placed over the temporal bone after removal of part of the temporalis muscle; relative blood flow laser Doppler flow probe during MCA occlusion (open circles) and 30 minutes after reperfusion (filled circles) in animals receiving vehicle, 0.5 mg/kg or 1 mg/kg FTY720 (i.p.) are shown in panel A (n=12 for vehicle, n=6 for FTY720). In a separate cohort of mice, we used laser speckle flowmetry to study the spatio-temporal characteristics of CBF changes during focal cerebral ischemia in mice pre-treated with either saline (open circles) or FTY720 (1 mg/kg, i.p.; filled circles) 1h before distal middle cerebral artery occlusion. Laser speckle imaging was started 1 min before distal middle cerebral artery occlusion and continued throughout the experiment. Ischemic CBF deficit was analyzed over time by quantifying the area of cortex with either severe (0% to 20% residual CBF, shown in panel B) or moderate CBF reduction (21% to 30% residual CBF, shown in panel C). 60 minutes after distal MCA occlusion, the area of severely ischemic cortex (i.e. with ≤20% residual CBF) was 3.7±1.0mm² in FTY-treated mice (n=4), compared to 4.2±0.5 mm² in the saline-treated group (n=4) (Figure 1B); the area of mildly ischemic cortex was 6.3±0.5 mm² in FTY-treated mice, compared to 6.9±1.3 mm² in saline-treated mice (Figure 1C).

Figure 2

Effect of FTY720 post-treatment on infarct size, neurological deficit, and edema following 90-min MCAo in mice. A: Mice treated at reperfusion and at 24 hrs with saline (n=12), 0.5 mg/mg (n=5) or 1 mg/kg FTY720 (n=7) (i.p.) were decapitated after 48 hours. Infarct area was measured on six hematoxylin-stained frozen sections. Both doses of FTY720 significantly reduced infarct size. Only the higher dose improved neurological deficit assessed just before sacrifice (panel B). C: for assessment of brain edema, a separate cohort of mice (n=6 per group) was treated at the time of reperfusion with saline (open bars) or 1 mg/kg FTY720 (solid bars). Brain edema was assessed at 24 hours post reperfusion by measuring brain water content using wet weight minus dry weight method. FTY720 treatment significantly reduced the increase of brain water content to control levels in the ischemic hemisphere.

Figure 3

Effect of FTY720 treatment on cells death in vivo and in vitro. Mice were treated at reperfusion (following 90-min MCAo) and at 24 hrs with saline or 1 mg/kg FTY720 (i.p.) (n=7/group) and were decapitated after 48 hours. Analysis of cells exhibiting DNA fragmentation was performed using a fluorescein-based terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) to label double-stranded DNA breaks. TUNEL-labeled cells were counted in randomly selected fields of view by an investigator blinded to the treatment groups. For detailed analysis of the distribution of immunoreactive cells, we divided the coronal brain sections into ischemic core, peri-infarct area, ipsilateral intact area and contralateral area. A shows the distribution of counting fields ipsilateral to the lesion in a representative coronal brain section. B show representative TUNEL staining pattern and intensity in ipsilateral intact areas, the peri-infarct areas and cores of a vehicle- (top) and an FTY720-treated mouse (bottom). C: Quantitative analysis shows that FTY720 (1 mg/kg, i.p.; solid bars) significantly reduced the number of TUNEL positive cells in the periinfarct area (140±17 vs. 324±47 cells / mm2) and ischemic core (100.6±9 vs. 230.2±48 cells / mm2) when compared to vehicle-treated group (open bars; p < 0.05). D: Primary cortical neurons were exposed to 100 µM Glutamate for 5 minutes (open bars) or to 30 µM H₂O₂ for 30 minutes (solid bars). Cells were then treated with culture medium alone, or medium supplemented with various concentrations of FTY720 or S1P for 24 hours. Cells were then stained with Calcein AM, fixed in 4% PFA, and stained with DAPI. Healthy-looking cells were counted in three random fields by an investigator blinded to the treatment groups. Both Glu and H₂O₂ treatment reduced cell viability to about 25%. Neither FTY720 nor S1P treatment was able to rescue these cells (as a positive control, 100 µM z-DEVD-FMK significantly increased the number of healthy-looking cells, compared to vehicle-treated cells in both Glu and H₂O₂-induced toxicity experiments, p<0.05; 4 independent experiments performed in triplicate). Neuronal viability assays after H₂O₂ exposure using the MTT and LDH assays: neurons were exposed to H₂O₂ for 30 min as described in Supplemental Methods. Vehicle, S1P, FTY720 or phospho-FTY720 was then added to the cultures. Cell viability was assessed after 24 hours using the MTT (E) and LDH (F) assays as described in Supplemental Methods. None of the FTY720 or S1P concentrations tested were able to rescue neurons from H₂O₂-induced cell death.

Figure 4

Effect of FTY720 treatment on endothelial expression of ICAM-1 in vivo and in vitro. A: representative ICAM-1 staining pattern and intensity in two peri-infarct and two core areas of a vehicle- (top) and an FTY720-treated mouse (bottom). Mice were treated at reperfusion (following 90-min MCAo) and at 24 hrs with saline or 1 mg/kg FTY720 (i.p.) and were decapitated after 48 hours (n=7/group). B: the length of vessels stained by the ICAM-1 antibody per area was measured by an investigator blinded to the treatment groups as described in Methods. This length was significantly shorter in the periinfarct (298.6±43 vs. 583.93±83 µm / mm²), ipsilateral intact (3.94±1 vs. 29.26±8 µm / mm²) and contralateral areas (3.61±1 vs. 8.87±1 µm / mm²) of FTY720-treated mice (solid bars) than saline-treated mice (open bars). C: Human brain microvascular endothelial cells treated in vitro with tumor necrosis factor (TNF-α) showed enhanced ICAM-1 protein expression as shown on this representative Western blot (see panel D for conditions). D: Western blots were quantified by image analysis: FTY720 (0.6 µM) and S1P (1 µM) significantly reduced ICAM-1 up-regulation in endothelial cells by approximately 50%; there was no effect of 4 mg/ml BSA treatment (S1P vehicle) (4 independent experiments).

Figure 5

Effect of FTY720 treatment of the expression of inflammatory markers 48 hrs after 90-min MCAo. Mice were treated at reperfusion (following 90-min MCAo) and at 24 hrs with saline (open bars) or 1 mg/kg FTY720 (i.p.) (solid bars) (n=7/group). Immunohistochemistry and cell counting were performed as described in Methods. A: FTY720 treatment (1 mg/kg, i.p.) significantly decreased the number of cells immuno-reactive for myeloperoxidase (MPO, a marker enzyme for neutrophils) in all the areas: periinfarct area (25±4 vs. 48±4 cells / mm²), ischemic core (16±3 vs. 25±2 cells / mm²), ipsilateral intact area (3±0.3 vs. 4±0.4 cells / mm²) and contralateral area (2.3±0.3 vs. 4.3±0.3 cells / mm²) (p < 0.05). B: Mac-1 is an adhesion protein involved in neutrophil extravasation during inflammation, its expression and activity are greatly increased after neutrophil activation. FTY720 significantly decreased the number of Mac-1 labeled cells in the periinfarct area (24±3 vs. 37±4 cells / mm²) and ischemic core (18±2 vs. 27±3 cells / mm²). Iba1 is specifically expressed in macrophages/microglia and is upregulated during the activation of these cells. Microglia were considered activated when they exhibited stout, partially retracted processes extending from the cell perikarya. C: FTY720 treatment decreased the number of Iba1-positive cells with an “activated morphology” in the periinfarct area (162±13 vs. 223±14 cells/mm²), ipsilateral intact area (131±20 vs. 193±10 cells / mm²) and ischemic core (7±4 vs. 42±11 cells / mm²), but did not affect the number of Iba1-positive cells exhibiting a resting morphology (D).

Figure 6

Long term effects of FTY720 treatment and effect in other rodent stroke models. Mice received 3 mg/kg FTY720 2 hours after reperfusion (following 90-min MCAo), at 24 hours and once again at 48 hours. Neurological deficit and motor function in the wire grip test were assessed at day 1, 3, 7, 10 and 14 (baseline wire grip performance was also assessed the day before MCAo). A: There was an overall statistically significant difference in neurological deficit score between vehicle- (open circles; n=10) and FTY720-treated mice (filled circles; n=8) (p<0.001, Friedman repeated measures ANOVA on ranks). When days were analyzed individually, there was a difference between vehicle- and FTY720-treated mice on day 1 (p=0.01), day 10 (p = 0.02), and day 14 (p = 0.006), but not on day 0, 3 and 7 (Mann-Whitney Rank Sum Test). B: Compared with vehicle-treated mice (open circles), wire-grip test performance was significantly improved in FTY720-treated mice (filled circles) over the experimental period (p<0.05); when days were analyzed individually, there was a difference between vehicle- and FTY720-treated mice on day 10 (p=0.05) and 14 (p=0.04). C: Sprague-Dawley rats underwent 2-hour MCAo as described in Methods. FTY720 (1 mg/mg, administered i.p. 30 minutes after reperfusion) significantly decreased infarct volume measured 22 hours after reperfusion (n=9/group). D: In a permanent mouse MCAo model, 1 mg/kg FTY720 significantly decreased infarct size when administered either 2 or 4 hours after the beginning of the occlusion (compared to time-matched saline-treated mice) (n=9/group).