Absence of perforin expression confers axonal protection despite demyelination

Abstract

Current evidence suggests that demyelination may be a necessary but not a sufficient condition for neurologic deficits associated with multiple sclerosis. Axon injury that occurs within the permissive environment of the demyelinated lesion is better correlated with functional deficits, but the mechanisms and cellular effectors of this injury are largely unknown. In an effort to identify potential axon injury mediators, we examined demyelination, motor function, and the number of spinal axons in perforin-deficient mice. Perforin is a critical molecular mediator of cytotoxic immunological injury and we hypothesized that genetic deletion of perforin expression would protect demyelinated axons. Indeed, we found that while perforin-deficient mice had considerable spinal cord demyelination 180 days after infection with Theiler's murine encephalomyelitis virus, such mice exhibited functional and axonal preservation comparable to non-demyelinated perforin-competent controls. We conclude that perforin-dependent effector cells such as cytotoxic T cells, gammadelta T cells, and natural killer cells may play a role in axon damage that is dependent upon but separable from demyelination.

Figure 1

Perforin-deficient mice exhibit substantial spinal cord demyelination 180 days after infection with TMEV. A) Perforin-competent C57BL/6J mice exhibit normal spinal cord myelination. B) Perforin-deficient C57BL/6J mice have regions of profound demyelination that are comparable to the demyelination observed in chronically-infected CD4-deficient mice (C). Scale bar in (C) is 50 μm and refers to all three panels. Images are representative of at least 8 mice per group and were collected in approximately the same region of cervical spinal cord.

Figure 2

Perforin-deficient mice exhibit preservation of function despite significant demyelination. Mice from all three groups were assessed by rotarod 180 days after infection. Functional performance of the perforin-deficient C57BL/6J mice (78.2 ± 7.8% of baseline, n=15) was indistinguishable from wildtype, perforin-competent C57BL/6J mice (83.2 ± 5.3% of baseline, n=9) and was significantly better than the performance measured in demyelinated CD4-deficient mice (43.8 ± 11.4% of baseline, n=7; P<0.009 vs. PFP^-/- and P<0.007 vs. PFP^+/+). Thus, despite the presence of demyelination in perforin-deficient C57BL/6J mice that was comparable to that measured in CD4-deficient mice, the absence of perforin expression conferred protection of motor function.

Figure 3

Perforin-deficient mice exhibit preservation of large diameter axons in the spinal cord despite significant demyelination. Mice from all three groups were morphologically assessed for the number of small (area = 1-4 μm²), medium (area = 4-10 μm²), and large (area ≥ 10 μm²) axons in the mid-thoracic spinal cord. Values are shown as number of axons per mm² analyzed. Perforin-deficient and perforin-competent wildtype mice were not different with regard to the number of large axons (see text), but such axons were reduced in CD4-deficient mice. On the other hand, the number of small and medium axons was reduced in both perforin-deficient and CD4-deficient mice as compared to wildtype mice. The functional preservation observed in perforin-deficient mice relative to CD4-deficient mice suggests that protection of large axons is sufficient to maintain motor function and also suggests that perforin directly injures large spinal axons during demyelination.