Simvastatin inhibits remyelination in cuprizone-demyelinated corpus callosum. A: Luxol fast blue (LFB) histochemistry (left panels) and myelin basic protein (MBP) immunohistochemistry (right panels) were used to evaluate myelin content in the corpus callosum (CC). Representative images of sections of animals treated with vehicle (Veh) or simvastatin (Simva) for weeks 4 to 9 or weeks 7 to 9. Scale bar = 200 μm. B: Average LFB demyelination scores (± SEM) for animals treated with vehicle or simvastatin during weeks 4 to 6. By week 6, simvastatin treatment of animals on normal diet (black bars) had no effect on myelin load relative to vehicle control. Cuprizone administration (gray bars) significantly increased demyelination score relative to vehicle-treated animals on normal diet (black bars). Simvastatin treatment of cuprizone-fed animals further increased demyelination scores. C: Average LFB demyelination scores (± SEM) for animals treated with vehicle or simvastatin for weeks 4 to 9 or 7 to 9. Long-term simvastatin treatment of animals on normal diet (black bars) significantly increased demyelination score. Cuprizone treated animals (gray bars) injected with vehicle still demonstrated an overall increase in demyelination at week 9 relative to control, yet some recovery was observed as compared with week 6 (B). Simvastatin treatment (weeks 4 to 9, 7 to 9) produced persistent increases in demyelination scores relative to cuprizone alone. D: Average area of MBP staining in the CC (μm²± SEM) for animals treated with vehicle or simvastatin during weeks 4 to 9 or 7 to 9. Simvastatin treatment of animals on normal diet (black bar) caused a significant reduction in area of MBP staining relative to vehicle control. By week 9, MBP levels in the CC of animals treated with cuprizone (gray bar) and vehicle had recovered to those observed in animals on normal diet. Simvastatin treatment (weeks 4 to 9, 7 to 9) of cuprizone-fed animals resulted in a reduced area of MBP staining relative to cuprizone alone. Analysis of variance P values <0.001 for LFB scores and 0.0019 for MBP quantification. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Simvastatin impacts OPCs in the cuprizone-demyelinated corpus callosum. A: Coronal sections of corpus callosum of animals sacrificed at week 6. OPCs were identified by strong immunostaining against Olig2 (left panels) and Nkx2.2 (right panels) (brown). Nuclei were labeled with hematoxylin (blue). Simvastatin treatment (weeks 4 to 6) of animals on normal diet caused an increase in Olig2^strong OPCs and Nkx2.2^strong OPCs relative to vehicle control. Cuprizone-treated animals demonstrated an increase in Olig2^strong cells but not Nkx2.2^strong OPCs at this time. These animals also demonstrated an increase in total nuclei in the corpus callosum relative to animals on normal diet, which represented astroglial and microglial infiltration (addressed in Figure 7). Simvastatin treatment of animals on a cuprizone diet during weeks 4 to 6 caused a relative decrease in Olig2^strong and Nkx2.2^strong OPCs relative to cuprizone-vehicle controls. Scale bar = 50 μm. B: Coronal sections of corpus callosum of animals sacrificed at week 9. Simvastatin treatment of animals on normal diet had no effect on numbers of Olig2^strong OPCs but caused an increase in numbers of Nkx2.2^strong OPCs relative to vehicle controls. Cuprizone-treated animals showed a decrease in Olig2^strong OPCs and an increase in Nkx2.2^strong OPCs at week 9 relative to week 6. However, simvastatin treatment during weeks 4 to 9 or 7 to 9 caused a significant increase in Olig2^strong OPCs and decrease in Nkx2.2^strong OPCs relative to cuprizone alone. Scale bar = 50 μm.

Simvastatin inhibits the recovery of mature OLGs in the demyelinated corpus callosum. A: Coronal sections of corpus callosum of animals sacrificed at week 6. Mature OLG cell bodies were labeled against NogoA (brown) and counterstained with hematoxylin (blue). Simvastatin treatment (weeks 4 to 6) of animals on a normal diet caused a significant decline in NogoA+ mature OLGs. Cuprizone treatment was also associated with a decrease in NogoA+ mature OLGs relative to animals on normal diet. Simvastatin treatment during the period of initial OPC proliferation/maturation (weeks 4 to 6) caused a further reduction in NogoA+ cells. B: Coronal sections of corpus callosum of animals sacrificed at week 9. Animals on a normal diet treated with simvastatin demonstrated a decrease in NogoA+ OLGs relative to vehicle control. Simvastatin treatment during weeks 4 to 9 or 7 to 9 caused a significant decrease in mature OLGs relative to cuprizone-vehicle controls. Scale bar = 50 μm.

Treatment protocol. Cuprizone was administered for weeks 1 to 6 to induce demyelination in the corpus callosum (CC). A: OPC proliferation/maturation in the CC begins at week 4 of cuprizone treatment, during concomitant demyelination. Once animals are returned to normal diet (week 7), robust remyelination occurs (weeks 7 to 9, B). Simvastatin (2 mg/kg/day) was injected daily during the period of concomitant demyelination and OPC maturation (weeks 4 to 6, C), during the entire remyelination period (weeks 4 to 9, D) or during the remyelination only phase (weeks 7 to 9, E). As controls, animals on normal diet received simvastatin therapy from weeks 4 to 6 or 4 to 9.

Quantification of the impact of simvastatin on OPCs in the corpus callosum. A, B: Coronal sections of corpus callosum of normal animals on normal diet. OPCs (small arrowheads) were identified as having strong staining for the transcription factors Olig2 and Nkx2.2 and showed a random distribution pattern (brown). Weakly positive cells (large arrowheads) were taken to be mature OLGs and were aligned in rows. Nuclei were labeled with hematoxylin (blue). Scale bar = 25 μm. C: Average numbers of Olig2^strong OPCs per mm² medial corpus callosum (CC) ± SEM. Simvastatin treatment (Simva) (weeks 4 to 6) of animals on normal diet (black bars) caused a significant increase in Olig2^strong OPCs relative to vehicle control. Cuprizone administration (gray bars) resulted in an increase in Olig2^strong cells at this time. Simvastatin treatment during weeks 4 to 6 inhibited this increase in Olig2^strong OPCs in the CC. At week 9, vehicle treated animals on normal diet (black bars) show an increase in Olig2^strong cells compared with week 6. At this time point, simvastatin treatment of animals on normal diet had no significant effect on numbers of Olig2^strong OPCs. Cuprizone-treated animals showed a decrease in these cells by week 9 relative to week 6. However, simvastatin treatment during weeks 4 to 9 or 7 to 9 caused a significant increase in Olig2^strong OPCs in the CC relative to cuprizone alone. D: Average numbers of Nkx2.2^strong OPCs per mm² medial CC ± SEM. Simvastatin treatment (weeks 4 to 6) of animals on normal diet (black bars) caused a significant increase in Nkx2.2^strong cells in comparison with control (vehicle + normal diet). Simvastatin treatment (weeks 4 to 6) of animals on cuprizone (gray bars) was associated with a decrease in Nkx2.2^strong OPCs in the CC versus control (cuprizone + vehicle). By week 9, vehicle treated animals on normal diet (black bars) showed a decrease in Nkx2.2^strong cells compared with week 6. At this time point, simvastatin treatment of animals on normal diet caused an increase in numbers of Nkx2.2^strong OPCs. Cuprizone administration induced a significant increase in Nkx2.2^strong OPCs by week 9. Simvastatin treatment (weeks 4 to 9) of animals on cuprizone caused a significant decrease in numbers of Nkx2.2^strong OPCs in the CC relative to cuprizone alone. Analysis of variance P values <0.001. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Quantification of the effect of simvastatin on mature OLGs in the corpus callosum. A: Mature OLG cell bodies were labeled with a NogoA-directed antibody (brown) and nuclei were labeled with hematoxylin (blue). Scale bar = 50 μm. B: Average numbers of NogoA+ cells per mm² of the medial corpus callosum (CC) (± SEM). Simvastatin treatment (weeks 4 to 6; Simva) of animals on normal diet (black bars) caused a significant decline in NogoA+ mature OLGs in the CC. Cuprizone-induced demyelination (gray bars) was associated with a decrease in NogoA+ mature OLGs (week 6) relative to animals on normal diet. Simvastatin treatment during the period of initial OPC proliferation/maturation (weeks 4 to 6) caused a significant reduction in NogoA+ cells relative to control. Although numbers of NogoA+ cells were maintained over time in vehicle treated animals on normal diet (from weeks 6 to 9), animals treated with simvastatin had a persistent decrease in NogoA+ OLGs in the CC by week 9. The cuprizone-vehicle treatment group demonstrated a recovery of NogoA+ OLGs at week 9 relative to week 6. However, simvastatin treatment during weeks 4 to 9 or 7 to 9 caused a significant decrease in mature OLGs in the CC. Analysis of variance P values <0.001. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.

Simvastatin does not decrease gliosis in the cuprizone model. A: Representative images of sections immunostained against GFAP (astrocytes; brown) and counterstained with hematoxylin (blue). Scale bar = 200 μm. B: Average numbers of GFAP+ astrocytes per mm² of the corpus callosum (CC) ± SEM. Simvastatin treatment (weeks 4 to 6) of animals on normal diet caused an increase in astrocyte numbers relative to vehicle alone (black bars). Cuprizone administration (gray bars) increased astrocyte reactivity in the CC, which was not affected by concomitant simvastatin treatment. Long-term simvastatin treatment (weeks 4 to 9) of animals on normal diet showed no difference in astrocyte reactivity relative to vehicle alone. The cuprizone treated animals showed a decrease in astrocyte numbers by week 9 relative to week 6; similar profiles were observed when simvastatin was administered to cuprizone-fed animals. C: Average numbers of IBA-1+ microglia/macrophages per mm² of the CC ± SEM. Simvastatin treatment (weeks 4 to 6) of animals on normal diet (black bars) had no significant effect on IBA-1+ cell number in the CC. By week 6, cuprizone administration (gray bars) caused an increase in IBA-1+ cell numbers relative to vehicle controls; this was not affected when simvastatin was administered. Long-term simvastatin treatment (weeks 4 to 9) of animals on normal diet was associated with an increase in microglia reactivity relative to control. At the end of the recovery from cuprizone-induced demyelination, microglia numbers were further increased relative to vehicle control. Simvastatin treatment of cuprizone-fed animals during weeks 4 to 9 or 7 to 9 had no effect on IBA-1+ cell numbers relative to cuprizone controls. Analysis of variance P values <0.001. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.