Pathological inflammation and dendritic beading in Lewis rat LSSC in acute EAE. A to C and D to F show H&E staining and MAP2 IHC in LSSC transverse sections obtained from normal, 12-dpi EAE peak, and 20-dpi EAE recovered animals, respectively. The lateral part of the ventral horn is shown on the left of each photograph, and the white matter lateral to the ventral horn is shown on the right. G to I and J to L show MAP2 staining in the gray matter (GM) and the white matter (WM) separately under higher magnifications. In normal LSSC sections, H&E staining shows a few large motoneurons and some other neural cells (A). MAP2 immunostaining strongly stained motoneuron perikarya and the intricate dendritic network in gray matter (D and G). The dendrites form transverse bundles at the gray and white matter junction area, and extensively penetrate into the lateral funiculus of the white matter (D and J). At the EAE peak, H&E staining (B) shows severe perivascular and parenchymal inflammation in LSSC. In the gray matter, MAP2 staining (E and H) shows that some proximal dendrites have mild swelling, but there is no dendritic beading. In contrast, most dendrites are beaded in the white matter (E and K). When EAE is recovered, inflammation (C) and dendritic changes (F, I, and L) resolve in both the gray and white matter, although some residual dendritic beading is still present in the white matter (L). Scale bars: 50 μm (A–F); 25 μm (G–L).

CTB retrograde labeling of L2-L4 spinal motoneurons in the acute EAE model. A, B, and C show gray matter labeling in normal, 12-dpi EAE peak and 28-dpi EAE recovered animals. The morphology of motoneurons and the gray matter dendrites do not show marked differences, and there was no dendritic beading at the EAE peak. D, E, and F show white matter dendritic labeling in normal, EAE peak, and recovered animals. White matter dendrites in normal spinal cord (D, arrowheads) are labeled as fine lines with fairly constant thickness. At the EAE peak (E), most white matter dendrites show a typical beading pattern, and the majority of swellings (arrowheads) are 10 to 20 μm in diameter. The intersegments, although continuous between beads, are thinner than normal dendrites. These changes resolve in most white matter dendrites (F) after clinical EAE recovery. Scale bars, 25 μm.

Pathological inflammation and dendritic beading in DA rat LSSC during chronic EAE. Please note that in this figure various EAE stages are arranged in columns while different staining is in rows. H&E staining (A–E) shows that perivascular and parenchymal inflammation are evident in both the gray and white matter of LSSC at the first EAE peak (B) and EAE relapse (D), but resolve at EAE remission (C) and after EAE recovery (E). Throughout the entire chronic EAE course, MAP2 IHC shows little morphological changes in the gray matter dendrites (F–J). In contrast, extensive dendritic beading occurs in the white matter at the first EAE peak (L) and EAE relapse (N), but is substantially recovered at EAE remission (M) and after clinical EAE recovery (O). Scale bars: 50 μm (A–E); 25 μm (F–O).

Quantitation of MAP2 IHC at different stages of acute EAE (A) and chronic EAE (B). Analysis of variance tests showed significant differences (P < 0.01) in all four groups of data, and post hoc tests were applied to examine the significance of differences between individual subgroups. Asterisks are shown above the bars when the mean values are significantly different (P < 0.05) from their immediate previous ones. Asterisks are also shown when the differences between the normal and recovery levels are statistically significant (P < 0.05). Results show that in the gray matter of LSSC, MAP2 signals are significantly reduced in acute EAE and in the first episode of chronic EAE. After EAE recovery, there are no significant differences in the gray matter in either EAE model. In white matter, signals of MAP2 IHC are markedly reduced in acute EAE, in the first episode and relapse phase of chronic EAE. There is significant recovery of white matter MAP2 signals during EAE recovery and remission. In the chronic but not acute EAE model, white matter MAP2 signals are significantly lower than normal after clinical EAE recovery.

Immunoblot analysis of MAP2 levels in LSSC at different stages of acute EAE [0.5⁰, 1⁰, 2⁰, 3⁰, and recovery (R)] compared with those in normal (N) LSSC. Different lanes represent protein samples from different animals. The amount of loaded protein was shown with β-actin immunoblot. The data show that MAP2 protein level in LSSC was markedly reduced during EAE, but was almost completely recovered after clinical EAE recovery.

The changes of synaptic protein immunostaining in the gray matter of LSSC in acute EAE. In the normal spinal cord (A–C), large motoneurons and their proximal dendrites in the ventral horns are positively stained in synaptophysin, synapsin I, and PSD-95 IHC. Punctated staining in thinner dendrites is also evident. At the EAE peak (D–F), the positive staining around neuronal somata as well as the overall gray matter staining is reduced. The synaptic protein signals are substantially recovered after clinical EAE recovery (G–I). Scale bars, 25 μm.

The changes of synaptic protein immunostaining in the white matter of LSSC in acute EAE. Synaptophysin, synapsin I, and PSD-95 IHC (A–C) all show branch-like patterns in the normal white matter, which are similar to the MAP2 staining pattern. At the EAE peak, signals for all three synaptic proteins are markedly reduced (D–F), while some inflammatory cells are positive for PSD-95 (F). Synaptic protein signals show partial recovery at 20 dpi when clinical EAE is fully recovered (G–I). Scale bars, 25 μm.

Quantitation of synaptic protein IHC in acute EAE. Both the area of positive staining (A) and the number of stained neurons (B) were quantitated from the ventral gray matter. The area of positive staining in the white matter was also quantitated (C). Analysis of variance tests showed that significant differences existed within all nine groups (P < 0.01), and post hoc tests were performed to examine the significance of differences between individual subgroups. Asterisks were shown in the same way as described in Figure 4 ▶ . The results show that positive signals for all three markers were significantly reduced in both the gray and white matter of LSSC at the acute EAE peak. Significant recovery in these synaptic protein signals was found after clinical EAE recovery. However, except for the gray matter area positive in synaptophysin and synapsin I IHC, other measurements only show partial recovery at 20 dpi.

Immunoblot analysis of synaptophysin, synapsin I, and PSD-95 levels in LSSC at different EAE stages [0.5⁰, 1⁰, 2⁰, 3⁰, and recovery (R)] compared with those in normal (N) LSSC. Different lanes represent protein samples from different animals. The amount of loaded protein was shown with β-actin immunoblot. The two bands in synapsin I immunoblot represent the 80 kd of synapsin Ia and the 77 kd of synapsin Ib. The marked reduction in synaptophysin and synapsin I immunoreactivity was well correlated with EAE severity. The reduction in PSD-95 level was strong even at the EAE onset. There was marked recovery of immunoreactivity for these synaptic proteins on clinical EAE recovery at 20 dpi.

Quantitation of synaptic protein IHC in chronic EAE. Analysis of variance tests showed that significant differences existed within all nine groups (P < 0.01), and the pairwise data are presented in the same way as in Figure 7 ▶ . The results show that the reduction of synaptic protein signals was significant in the first EAE episode and during EAE relapse, and that the recovery of staining signals was significant in EAE remission and EAE recovery. The only exception is that there is no significant recovery of synapsin I-positive neuron numbers at EAE remission. Except for the gray matter area positive for synaptophysin and synapsin I, other measurements only show partial recovery 12 days after full clinical EAE recovery.