Figure 3.

Laquinimod (LQ) treatment attenuates inflammation and demyelination in spinal cords of EAE mice. (A) Consecutive Thy1-YFP (green) thoracic spinal cord sections co-immunostained with CD45 (red, i) or GFAP (red, ii) at 10× magnification are shown from partial images (showing dorsal column; DC) from normal control, vehicle-treated EAE, 5 mg/kg LQ pretreated EAE, and 25 mg/kg LQ pretreated EAE mice at day 34. Spinal cords from vehicle-treated EAE mice show large areas of CD45+ and GFAP+ cell staining in the dorsal column as compared with normal controls, whereas 5 mg/kg and 25 mg/kg LQ pretreated EAE mice show only occasional CD45 and GFAP positivity. Consecutive sections from the same mice were also scanned at 40× magnification (within DC; designated by the area within the dotted line square) to show the morphology of CD45+ and GFAP+ cells. Number of CD45+/DAPI+ (iii), CD3+/DAPI+ (v), and GFAP+/DAPI+ (vi) cells per 400 μm² within the DC were quantified from consecutive immunostained sections. Vehicle-treated EAE mice had a significant increase in CD45, CD3, and GFAP cells compared to healthy controls, whereas 5 mg/kg and 25 mg/kg LQ pre- and early post-treated EAE mice did not. A coronal cross section of thoracic spinal cord from a Thy1-YFP (green) and DAPI-stained (red) vehicle-treated EAE mouse imaged at 4× magnification (iv) is shown. The various regions of the spinal cord section are labeled as: DC, dorsal column; LF, lateral funiculus; VF, ventral funiculus; GM, gray matter. Statistically significant compared with normal controls (*P < 0.05; **P < 0.001; 1 × 4 ANOVAs; n = 6–8 mice/group). Data are representative of experiments repeated in their entirety on another set of EAE mice, with each of the treatment groups. (B) At post-immunization day 34, vehicle-treated EAE mice had reduced MBP (i), increased APP (ii), and decreased MBP+NF200 (iii) immunoreactivity, compared to normal controls, in the DC and VF of thoracic spinal cord sections imaged at 10× (i) and 40× (ii, iii) magnification. In contrast, 5 mg/kg and 25 mg/kg LQ pre- and early post-treated EAE mice showed relatively preserved MBP and NF200 staining. In addition, there was less APP+ immunoreactivity in LQ-treated groups as compared to vehicle-treated groups (ii). Upon quantification (iv–vi), MBP and NF200 immunoreactivity in the DC was significantly lower in vehicle-treated EAE mice as compared with normal mice, whereas LQ-treated EAE mice demonstrated no such significant decreases. Quantification revealed less MBP+ and NF200+ axons in the VF of vehicle-treated mice as compared to normal controls. LQ treatment reversed this trend; that is, there were less APP+ axons and more myelinated (MBP+ and NF200+) axons. Myelin density is presented as percent of normal. Statistically significant compared with normal controls (* P < 0.05; ** P < 0.001; 1 × 4 ANOVAs; n = 6–8 mice/group).