Comparison of Reported Spinal Cord Lesions in Progressive Multiple Sclerosis with Theiler’s Murine Encephalomyelitis Virus Induced Demyelinating Disease

Figures

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  Figure 1. Clinical investigation and rotarod testing of TMEV- and mock-infected SJL mice. TMEV-infected mice showed significant clinical signs starting at 91 dpi (A) and a deterioration of motor coordination starting at 63 dpi (B) compared to mock infected animals. Graphs display mean and standard error of mean. Significant differences between the groups as detected by two-way repeated measure ANOVA with post-hoc independent t-tests for the different time points are marked by asterisks (* p ≤ 0.05).

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  Figure 2. Assessment of spinal cord areas revealed a significant reduction of cross-sectional area at 196 dpi within the thoracic segment (A), accompanied by a reduction of thoracic white matter area at 196 dpi (B) and a reduction of the area of rostral reticulospinal tract (rrts) within the thoracic segment at 98 dpi and within the cervical and thoracic segment at 196 dpi (C). Graphs display box and whisker plots. Significant differences between groups detected by Mann–Whitney U-test were indicated by asterisks (* p ≤ 0.05), GM = gray matter, WM = white matter.

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  Figure 3. Evaluation of infiltrating macrophages within the area of the rostral reticulospinal tract revealed a significant increase in Mac3+ cells within the cervical segment at 42 dpi, within the cervical and thoracic segment at 98 and 147 dpi as well as within all investigated segments at 196 dpi (A). Assessment of myelin basic protein (MBP) stained slices revealed a significantly reduced MBP positive white matter (WM) area in the thoracic segment at 42 dpi and in all investigated segments at 98, 147, and 196 dpi (B). Moreover, the thoracic segment showed a significant reduction of MBP positive area in the gray matter (GM) at 98 dpi (C). No significant differences between TMEV-infected groups comparing 147 and 196 dpi were detected. Graphs display box and whisker plots. Significant differences between groups detected by Mann–Whitney U-test were indicated by asterisks (* p ≤ 0.05).

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  Figure 4. Evaluation of axonal loss showed a decreased number of phosphorylated neurofilaments (pNF) positive small (1–4<μm) diameter axons within the thoracic and cervical spinal cord at 147 dpi and within the cervical segment at 196 dpi (A). Large (≥4 μm) axons were significantly decreased within the thoracic segment at 98 dpi (B). No significant differences between TMEV-infected groups comparing 147 and 196 dpi were detected. Graphs display box and whisker plots. Significant differences between groups detected by Mann–Whitney U-test were indicated by asterisks (* p ≤ 0.05).

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  Figure 5. Ultrastructural analysis of degenerative changes of axons and myelin sheaths of TMEV infected SJL-mice. (A) Demyelinated area with denuded axons [arrowheads] and infiltration of microglia/macrophages [M] and axonal degeneration [asterisk] within the ventrolateral areas of the spinal cord (TMEV infected animal, 42 dpi, magnification 5000×). (B) Vacuolation [V] of myelin sheath [S] surrounding an intact axon [A] (TMEV infected animal, 98 dpi, magnification 10,000×). (C) Axonal [A] degeneration characterized by shrinkage, an uneven axonal membrane and accumulation of electron dense material (TMEV infected animal, 98 dpi, magnification 5000×). (D) Multiple remyelinated axons [arrows], characterized by thinner myelin sheaths compared to normally myelinated fibers [triangles], characteristic for oligodendrocyte [O] mediated remyelination were detected (TMEV infected animal, 196 dpi, magnification 6300×).

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  Figure 6. Histochemical and immunohistochemical evaluation of SC segments using Luxol fast blue (LFB) staining (A: mock infected animal showing regularly myelinated white matter; B: TMEV infected animal with multifocal areas of demyelination). Immunohistochemistry targeting myelin basic protein (MBP; C: mock infected animal showing regularly myelinated white matter; D: TMEV infected animal with multifocal demyelination), phosphorylated neurofilaments (pNF; E: mock infected animal showing regularly distributed pNF labeled axons; F: TMEV infected animal with multifocal loss of pNF labeled axons) and microglia/ macrophages (Mac3; G: mock infected animal showing no immunolabeled cells; H: TMEV infected animal revealing multifocal immunolabeled cells within the white matter). Pictures show the thoracic segment of a mock (clinical score: 0, rotarod: 27.8 rpm) and a TMEV-infected animal (clinical score: 4; rotarod: 14.5 rpm) at 98 dpi. Scale bars = 200 μm. Inserts in 400× magnification.

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  Figure 7. Quantification of cross-sectional area (CSA; black line), white matter (WM), gray matter (GM; blue line) and the approximated area of the rostral reticulospinal tract (rrts; red dashed line) using MBP-stained sections. Localization of rrts is marked as gray area. Area of rrts was approximated via an angle bisector (dashed line) between a vertical and horizontal line intersecting at the central canal (matrix: A; application example: B) For assessment of axonal loss and inflammation, images from the rrts were taken at high magnification (40× objective). One picture was taken from the left and right ventromedial white matter each, saved as tiff file (C) and a counting frame (120 μm × 120 μm) was inserted (D). For quantification of axonal density, images were converted to 8bit grayscale (D). After an adjustment of threshold axons were counted and separated in small (1–<4 μm2) and large axons (≥4 μm2) via adjustment of size of the detected particles. Axonal density was calculated as pNF+ particles per mm2. Mac3+ cells within the counting frame were counted manually and calculated as Mac3+ cells per mm2 (D).