Figure 1.
EAE induction in C57/BL6 mice by adoptive transfer of encephalitogenic cells (at-EAE) or active immunization (active EAE).
Incidence in at-EAE mice (A) and active EAE mice (B) between day 7 and 15 after immunization or adoptive transfer. (A) at-EAE symptoms appeared on day 8 after immunization and the 100% of incidence is reached on day 14. (B) Active EAE symptoms appeared on day 10 after cells injection and the 100% of incidence is reached on day 15. (C) Average score of at-EAE and active EAE from the day of immunization or adoptive transfer to day 19. Increasing the score in at-EAE mice and active EAE is significant with respect to control mice (at-EAE: day 11, *, p<0.05; day 12, *, p<0.05; day 13, **, p<0.01; day 14, ***, p<0.001; day 15, **, p<0.01; day 17, **, p<0.01; day 18, **, p<0.01; 19, *, p<0.05; active EAE: day 18, *, p<0.05; day 19, *, p<0.05). After immunization or adoptive transfer score is significantly higher in at-EAE than in active EAE (day 10, ##, p<0.01; day 11, ###, p<0.001; day 12, ###, p<0.001; day 13 ##, p<0.01; day 14, ##, p<0.01). (D) Weight variations during EAE development. Weight loss in at-EAE mice is significant with respect to control mice between days 14 and 19 (day 14, **, p<0.01; day 17, **, p<0.01; day 18, **, p<0.01; and day 19, **, p<0.01). Significant weight differences are observed between at-EAE and active EAE on day 2 (###, p<0.001), day 7 (#, p<0.05) and day 12 (#, p<0.05). Data are representative of three separate experiments. Each group represents the mean of 6 mice for control, 13 mice for both at-EAE and active EAE, with SEM error bars. All statistical analyses were performed using Kruskal-Wallis nonparametric test followed by Dunn’s post analysis. Values of p < 0.05 were considered statistically significant.
Table 1.
at-EAE versus active EAE models.
Figure 2.
Glial activation in lumbar spinal cord.
Astroglial GFAP immunostaining is observed in lumbar spinal cord of at-EAE mice (B) and active EAE mice (C) when compared with control (A). Isolectin labeling in the lumbar spinal cord show increase in at-EAE mice (E) compared with active EAE (F) and control mice (D). Iba-1 immunostaing (microglial marker) is up-expressed in the lumbar spinal cord of at-EAE mice (H) compared with active EAE (I) and control mice (G). Semiquantitative histological analysis of glial markers in lumbar spinal cord (J) and in ventricular regions of brain (K). A significant increase of glial-positive cells is observed in at-EAE versus control both in lumbar spinal cord (GFAP: *, p<0.05; Isolectin and Iba-1: **, p<0.01) and brain (GFAP and Isolectin: *, p<0.05). The increased of Isolectin-positive cells is significantly higher in at-EAE than in active EAE (#, p<0.05). Data are representative of three separate experiments. Each group represents the mean of 4 mice for control, 10 mice for at-EAE and 7 mice for active EAE, with SEM error bars. All statistical analyses were performed using Kruskal-Wallis nonparametric test followed by Dunn’s post analysis. Values of p < 0.05 were considered statistically significant. Magnification bar 150 µm.
Figure 3.
Demyelination and neurodegeneration markers in the lumbar spinal cord.
The demyelination is clearly demonstrated by a loss of signal in the luxol fast blue staining (A–C) and MOSP (D–F) and MBP (G–I) immunostaining of at-EAE (B, E and H, respectively) and active EAE mice (C, F and I, respectively) when compared with control mice (A, D and G, respectively). NeuN immunostaining in control (J), at-EAE mice (K) and active EAE (L). None FluoroJade staining is observed in control mice (M), while some FluoroJade positive cells are observed in at-EAE mice (N) and few positive cells are observed in active EAE mice (O). Semiquantitative histological analysis of demyelination (P) and neurodegeneration (Q) markers in lumbar spinal cord. A significant decrease of immunostaining is observed in at-EAE versus control in lumbar spinal cord (MOSP and MBP: *, p<0.05). The decrease of MOSP immunostaining in at-EAE mice is significant with respect to the active EAE mice (#, p<0.05). No changes are detected in the NeuN-positive cell number meanwhile an increase in the number of degenerating neurons (FluoroJade) is observed in at-EAE mice with respect to control mice (**, p<0.01). Data are representative of three separate experiments. Each group represents the mean of 4 mice for control, 10 mice for at-EAE and 7 mice for active EAE, with SEM error bars. All statistical analyses were performed using Kruskal-Wallis nonparametric test followed by Dunn’s post analysis. Values of p<0.05 were considered statistically significant. Magnification bar 200 µm.
Figure 4.
Lymphocyte infiltrates in lumbar spinal cord.
H/E technique show absence of blood infiltrates in control mice (A) while many infiltrates are observed in at-EAE (B) and active EAE mice (C). No T cells (CD3 marker) immunostaining is observed in lumbar spinal cord of control mice (D). Many T cells are detected in the parenchyma of at-EAE mice and active EAE (E and F, respectively). No B-cells (CD-45R marker) immunostaining is observed in lumbar spinal cord of control mice (G). Many B cells are observed mainly in at-EAE mice (H) while few B-cells are detected in active EAE (I). Semiquantitative histological analysis of infiltration markers in lumbar spinal cord (J) and brain (K). A significant increase of CD-45R-positive cells is detected in at-EAE mice with respect to control mice both in lumbar spinal cord (**, p<0.01) and brain (**, p<0.01). The increase of CD-45R-positive cells was higher in at-EAE than in active EAE , both in lumbar spinal cord (#, p<0.05) and in brain (##, p<0.01). An increase in the number of CD3-positive cells has been observed in at-EAE in both CNS areas (**, p<0.01). Data are representative of three separate experiments. Each group represents the mean of 4 mice for control, 10 mice for at-EAE and 7 mice for active EAE, with SEM error bars. All statistical analyses were performed using Kruskal-Wallis nonparametric test followed by Dunn’s post analysis. Values of p < 0.05 were considered statistically significant. Magnification bar 150 µm.
Figure 5.
Double immunostaining for B cells in at-EAE and control spinal cord sections.
DAPI (A), CD-45R (B), IgG (C), merge (D) at-EAE fluorostaining sections, and control merge (E) fluorostaining sections. DAPI (F), CD3 (G), CD-45R (H), merge (I) at-EAE fluorostaining sections, and control merge (J) fluorostaining sections. DAPI (K), CD-138 (L), IgG (M), merge (N) at-EAE fluorostaining sections, and control merge (O) fluorostaining sections. DAPI (P), CD-138 (Q), CD-45R (R), merge (S) at-EAE fluorostaining sections, and control merge (T) fluorostaining sections. Magnification bar 150 µm.