Figure 4.

Proliferation of newly generated neurons visualized by immunostaining for the proliferation marker BrdU (red) and the immature neuronal marker DCX (green) in the neuroproliferative zones of C57BL/6 mice. Increased expression of BrdU and DCX in EAE mice and, to a greater extent, in EAE+GA mice in the SVZ (A; confocal images) and in the hippocampus (C) 25 d after EAE induction 1 d after the last GA injection. Right panels are high magnifications of the SGZ and the GCL. Note the DCX⁺ cells in the hippocampus that migrated into the GCL and manifest a dense and branched dendritic tree. B, DCX expression in the SVZ at different times points: 1 d (I), 10 d (II), and 30 d (III) after the last GA injection. Neuroproliferation declined with time; however, DCX expression in GA-treated mice was higher than that in EAE mice 1 and 10 d after treatment (coronal sections). Scale bar: A, 50 μm; B, C, 200 μm; C, right, 20 μm. st, Striatum; LV, lateral ventricle; IML, inner molecular layer; OML, outer molecular layer. D, Quantitative analysis of BrdU incorporation and DCX expression in EAE (red) and EAE+GA (blue) mice at various time points after EAE induction and GA treatment. Increased neuronal proliferation is observed in both neuroproliferative zones after disease appearance; subsequent decline is observed below that of naive mice and augmentation of neuroproliferation by the various schedules of GA treatment. Quantification was performed in the SVZ by counting BrdU-positive cells (Figure legend continues.)(Figure legend continued.) (those with BrdU/DCX dual staining) and measuring the DCX-stained area, starting at the level of the medial septum and 640 μm backward, and in the hippocampal DG by counting BrdU⁺/DCX⁺ cells (in both blades) and DCX⁺ cells (in the upper blade of the dentate) through its septotemporal axis. The number of BrdU/DCX-stained cells for each brain structure was averaged from eight unilateral levels per mouse (80 μm apart; 3–4 mice for treatment group). Results are expressed as change fold from naive controls. Control values for BrdU incorporation are as follows: SVZ, 211 ± 31 and 23 ± 6; hippocampus, 45 ± 13 and 17 ± 8; BrdU/DCX⁺ cells, 1 d and 1 month after the last BrdU injection, respectively; DCX staining, in the SVZ, 19, 464 ± 3550 μm², in the hippocampus, 78 ± 12 -positive cells averaged from 10 naive mice. Statistical analysis was performed by ANOVA followed by Fisher's LSD when appropriate. The asterisk indicates a significant effect over naive control and a significant effect over EAE-untreated mice (p < 0.05). E, Schedule of experiments: time length from EAE induction (day 0) until perfusion; GA injections as prevention (P), suppression (S), or delayed suppression (DS) treatments and BrdU inoculation, concurrently or immediately after GA treatment.