Axonal loss results in spinal cord atrophy, electrophysiological abnormalities and neurological deficits following demyelination in a chronic inflammatory model of multiple sclerosis

Abstract

Recent pathological studies have re-emphasized that axonal injury is present in patients with multiple sclerosis, the most common demyelinating disease of the CNS in humans. However, the temporal profile of demyelination and axonal loss in multiple sclerosis patients and their independent contributions to clinical and electrophysiological abnormalities are not completely understood. In this study, we used the Theiler's murine encephalomyelitis virus model of progressive CNS inflammatory demyelination to demonstrate that demyelination in the spinal cord is followed by a loss of medium to large myelinated fibres. By measuring spinal cord areas, motor-evoked potentials, and motor coordination and balance, we determined that axonal loss following demyelination was associated with electrophysiological abnormalities and correlated strongly with reduced motor coordination and spinal cord atrophy. These findings demonstrate that axonal loss can follow primary, immune-mediated demyelination in the CNS and that the severity of axonal loss correlates almost perfectly with the degree of spinal cord atrophy and neurological deficits.

Fig. 1

Demyelination in the spinal cords of susceptible SJL/J mice. (A) Following TMEV infection of susceptible SJL/J mice at 45 d.p.i. (closed circles) to 92–100 d.p.i. (closed triangles), demyelination progresses from 3 to 11%. The 14% of spinal cord white matter showing demyelination at 195–220 d.p.i. (open squares) was not statistically different from the demyelination observed at 92–100 d.p.i. The black line denotes the mean for the group. Each point represents the cumulative demyelination score for a single animal. Representative examples of thoracic spinal cord sections (B, C and D) are shown for an uninfected (B), a 45-day-infected (C) and a 195-day-infected (D) SJL/J mouse. The lesion size became progressively larger between 45 d.p.i. (C) and 195 d.p.i. (D); however, the lesion size at 92–100 and 195–220 d.p.i. was comparable. A section without demyelination (B) is shown for comparison. (Statistical differences were detected by one-way ANOVA. Pairwise comparisons were performed using the Student–Newman–Keuls method: P < 0.05.)

Fig. 2

Spinal cord atrophy in chronically infected SJL/J mice. Data are represented as the percentage change from a group of uninfected SJL/J mice. (A) Total cord atrophy was detected only in SJL/J mice infected for 195–220 days (n = 8), as indicated by the significant decrease from uninfected (n = 7) SJL/J mice. No significant decreases were observed in 195 d.p.i. C57BL/10 (n = 8) mice or 45 d.p.i. (n = 8) and 92–100 d.p.i. (n = 8) SJL/J mice. (B) Significant decreases in lateral and anterior white matter area were detected at C1–C7 and C8–T11 in 195–220 d.p.i. SJL/J mice. Atrophy was also observed at C8–T11 in 45 and 92–100 d.p.i. SJL/J mice; however, the atrophy was significantly less than that observed at 195–220 d.p.i. No decreases in area were detected for 195 d.p.i. C57BL/10 mice. (Asterisks denote statistical significance from uninfected SJL/J mice as detected by one-way ANOVA. Pairwise comparisons were performed using the Student–Newman–Keuls method: P < 0.05.)

Fig. 3

Frequency distributions of myelinated axonal areas in SJL/J mice. (A) No significant decreases in the relative frequencies of small myelinated axon fibres (0.1–4 μm²) were detected at 45 d.p.i. (n = 8), 92–100 d.p.i. (n = 8) or 195–220 d.p.i. (n = 8) compared with uninfected SJL/J mice (n = 7). (B) A significant decrease in medium to large myelinated fibres (4–10 μm²) was only detected in SJL/J mice infected for 195–220 days. (C) The largest myelinated axon fibres (≥10 μm²) were decreased by similar percentages at both 45 and 92–100 d.p.i. However, a marked decrease was observed at 195–220 d.p.i. (Asterisks denote a statistical difference from uninfected SJL/J mice as detected by one-way ANOVA. Pairwise comparisons were performed using the Student–Newman–Keuls method: P < 0.05.) (D) When the percentages of spinal cord demyelination were plotted against relative frequencies of medium to large myelinated fibres (≥4 μm²) for individual mice at 195–220 d.p.i., a strong negative correlation was observed (r = −0.84, P = 0.008). (E) A representative example of the sampling scheme used to calculate myelinated axonal area frequencies from the T6 spinal cord section of each animal. Areas with demyelination were excluded from the analysis. Eight 18 071 μm² fields (A, B, C, D, E, F, G and H) were captured in distinct anatomical regions of the T6 spinal cord white matter. (Boxes are drawn to scale.)

Fig. 4

Electrophysiological abnormalities in chronically infected SJL/J mice. (A) Comparable delays in the latency to the onset of MEPs were observed at 45 d.p.i. (n = 16) and 90 d.p.i. (n = 8) compared with uninfected (n = 22) SJL/J mice. A further delay was observed at 180 d.p.i. (n = 15). (B) Comparable reductions in the conduction velocities to the onset of MEPs were detected at 45 and 90 d.p.i. A significant reduction from these two time points was observed at 180 d.p.i. (C) The lowest mean amplitude (13 μV) was detected at 180 d.p.i. All other groups had amplitudes >17 μV. Representative tracings of MEPs are shown for an uninfected (D), a 45-day-infected (E) and a 180-day-infected (F) SJL/J mouse. Delays in the onset (arrows) of the MEPs were observed at 45 d.p.i. (E) compared with uninfected (D) SJL/J mice; however, amplitudes were preserved. Similar results were found at 90 d.p.i. Further delays in the latency to onset and reductions in amplitude were detected at 180 d.p.i. (F). (Conduction velocities and latencies between the groups were statistically compared using a one-way ANOVA. Pairwise comparisons were performed using the Student–Newman–Keuls method: P < 0.05. Amplitudes between 90 and 180 d.p.i. were compared using an unpaired Student’s t-test: P < 0.05.)

Fig. 5

Hierarchy of medium to large myelinated axon fibre loss in chronically infected SJL/J mice. Relative frequencies of large myelinated axon fibres (≥10 μrn²) are shown for uninfected (A), 45-day-infected (B), 92- to 100-day-infected (C) and 195- to 220-day-infected (D) SJL/J mice. Noticeable decreases in curve areas were detected at 45 and 92–100 d.p.i. However, the most significant reduction in curve area was noted at 195–200 d.p.i. (D). The hierarchy of medium to large myelinated fibre loss compared with uninfected SJL/J mice is illustrated for 45-day-infected (B inset), 92- to 100-day-infected (C inset) and 195- to 220-day-infected (D inset) mice. Ratios of infected/uninfected curve areas were calculated for all myelinated axonal area bins. Ratios equal to one signify preservation of a particular bin of axon fibres compared with uninfected SJL/J mice. Note the hierarchy of axonal loss at 45 dpi. (B inset) and 92–100 d.p.i. (C inset), with the largest fibres showing the most significant reductions. In contrast, SJL/J mice infected for 195–220 days were almost completely devoid of axons > 20 μm². Marked reductions were also apparent in all other medium to large axon size categories. Interestingly, at all time points, small axon fibres (0.1–5 μm²) were preserved.

Fig. 6

Correlations between pathological abnormalities and rotarod performance in SJL/J mice infected for 192 days. (A) A very strong positive linear correlation (r = 0.92, P = 0.008) was detected between me C7 combined lateral and anterior column area and rotarod performance in chronically infected (192 days) SJL/J mice. (B) A weaker negative correlation (r = −0.66, P = 0.11) approaching statistical significance was obtained between the percentage of spinal cord demyelination and rotarod performance. (C) Calculation of a correlation coefficient between the frequency of medium to large myelinated axon fibres (≥4 μm²) and rotarod performance revealed a strong positive linear relationship (r = 0.91, P = 0.01).