Demyelinated axons and motor function are protected by genetic deletion of perforin in a mouse model of multiple sclerosis

Abstract

Axon injury is a major determinant of the loss of neurological function in patients with multiple sclerosis. It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8 T cells and perforin in the Theiler murine encephalomyelitis virus model of multiple sclerosis have used mouse strains resistant to Theiler murine encephalomyelitis virus infection. To test the role of CD8 T cells in axon injury, we established a perforin-deficient mouse model on the H-2 major histocompatibility complex background thereby removing confounding factors related to viral biology in this Theiler murine encephalomyelitis virus-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2 mice, but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediator of axon injury and lend additional support to the hypothesis that CD8 T cells are primarily responsible for axon damage in multiple sclerosis.

Figure 1

Gene expression analysis in perforin-deficient mice. (A) Breeding strategy employed to generate the H-2^q perforin-deficient mice. (B) Experimental design utilized in this study. (C–F) Real-time RT-PCR analysis was performed on total RNA isolated from whole brain (C) or brain-infiltrating leukocytes (BILs) (D–F). (C) RNA was isolated from brain at 7 dpi and levels of GAPDH, granzyme B, and perforin were determined by RT-PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and granzyme B levels did not differ between the groups but perforin was not detected in perforin-deficient mice (n = 3 per group). Levels of gene expression are shown as mean change in crossing point relative to uninfected brain ± 95% confidence interval. (D) RNA was collected from BILs isolated at 7 dpi and levels of GAPDH, granzyme B, and perforin were determined by RT-PCR. GAPDH did not differ between the groups. There was a small but significant (p = 0.033; n = 3 per group) increase in granzyme B in the perforin-deficient BILs. Perforin was not detected in the perforin-deficient BILs. Levels of gene expression are shown as mean change in crossing point relative to background ± 95% confidence interval (uninfected mice do not have sufficient BILs for analysis). (E) Representative granzyme B amplicon melting curves and PCR products (inset) are shown for 3 mice in each group. (F) Representative perforin amplicon melting curves and PCR products (inset) are shown for 3 mice in each group. A perforin product was not observed in the perforin-deficient BILs. Expression differences were analyzed by t-test. IF = immunofluorescence; H&E = hematoxylin and eosin.

Figure 2

CD8⁺ cells infiltrate the spinal cord in chronically demyelinated mice irrespective of perforin expression. Spinal cord sections were prepared from perforin-competent (A–D) and perforin-deficient (E–H) mice at 180 dpi. (A–H) Representative inflammatory lesions in white matter are shown by hematoxylin and eosin stain (A, E). The lesions contain many cells that are immunopositive for CD8 in both perforin-competent (B, C) and perforin-deficient mice (F, G). Perforin co-staining (red) was observed in CD8⁺ cells (green) within demyelinated lesions in perforin-competent mice (D) but not in perforin-deficient mice (H); blue indicates DAPI. (I, J) Flow cytometric analysis at 180 dpi confirmed that the number of CD45^hiCD8⁺ cells present within isolated spinal cord infiltrating leukocytes did not differ between perforin-competent (I) and perforin-deficient mice (J). Scale bar in E = 50 µm and also refers to A. Scale bar in F = 50 µm and refers to B. Scale bar in G = 10 µm and refers to C. Scale bar in H = 1 µm and refers to D. dpi = days post-infection

Figure 3

Survival following Theiler’s murine encephalomyelitis virus infection is not altered by the absence of perforin. Survival was assessed through 180 days post-infection (dpi) in perforin-competent (solid grey line) and perforin-deficient (dashed line) mice. Two-way ANOVA revealed no statistically significant difference between the groups at any time point (p = 0.741; n = 15 per group at 0 dpi).

Figure 4

Spinal cord histopathology. Pathologic features in the spinal cord gray matter (A, B) and white matter (C–F) in perforin-competent mice (A, C, E) and perforin-deficient mice (B, D, F) at 7 days post-infection (dpi) (A, B), 45 dpi (C, D) and 180 dpi (E, F) in plastic-embedded sections stained with erichrome and cresyl violet. Acute neuronal disease, as suggested by neuronal swelling and inclusions and the proximity of numerous inflammatory cells is seen in both perforin-competent (arrowheads in A) and perforin-deficient (arrowheads in B) mice. Early demyelination (45 dpi) was morphologically similar between perforin-competent (C) and perforin-deficient (D) mice; arrowheads mark the general region of myelin damage. By 180 dpi, the appearances of the lesions (pale areas) did not differ between perforin-competent (E) and perforin-deficient (F) mice. Images were taken from mid-thoracic level and are representative of 10 mice in each group. Scale bar in D = 10 µm and refers to A–D. Scale bar in F = 50 µm and refers to E and F.

Figure 5

Neurologic function is preserved in perforin-deficient mice following Theiler’s murine encephalomyelitis virus infection and chronic demyelination. Rotarod performance was measured in perforin-competent (dark gray) and perforin-deficient (light gray) mice at 45, 90, and 180 days post-infection (dpi). Performance of 24 mice in each group that were followed longitudinally is shown as mean percent of baseline time-to-fall ± 95% confidence interval. Dead mice were retrospectively removed from the analysis; at 180 dpi, there were 19 perforin-competent and 22 perforin-deficient mice. Motor function was preserved in perforin-deficient mice throughout the course. P-values are derived from 2-way ANOVA.

Figure 6

Medium and large caliber spinal axons are preserved in perforin-deficient mice following Theiler’s murine encephalomyelitis virus (TMEV) infection and chronic demyelination. The absolute numbers of myelinated axon profiles were determined at the mid-thoracic level in the spinal cord of sham-infected wild type mice (black bars) and TMEV-infected perforin-competent (dark gray bars) and perforin-deficient (light gray bars) mice at 180 days postin-fection(dpi) (A). Axon counts are shown as mean ± 95% confidence interval derived from 20 sham, 16 perforin-competent, and 19 perforin-deficient mice. Small = 1–4 µm² area; medium = 4–10 µm² area; large = >10 µm² area. Representative images of mid-thoracic spinal cord araldite sections are shown at several magnifications for sham-infected (B, E, H, K, N) and TMEV-infected perforin-competent (C, F, I, L, O) and perforin-deficient (D, G, J, M, P) mice at 180 dpi. White boxes in B–J indicate the location of subsequent higher magnification panels. There is considerable loss of axons in both lateral (F, L) and ventral (I, O) white matter tracts in the perforin-competent animal. Preservation of medium and large axons is clear in the lateral (G, M) and ventral (J, P) cord in the perforin-deficient mouse. Scale bar in D = 200 µm and refers to B–D; scale bar in J = 100 µm and refers to E–J; scale bar in P = 25 µm and refers to K–P. P values derive from one-way ANOVA between strains for each axon size group.