Absence of neurological deficits following extensive demyelination in a class I-deficient murine model of multiple sclerosis

Abstract

Demyelination alone has been considered sufficient for development of neurological deficits following central nervous system (CNS) disease. However, extensive demyelination is not always associated with clinical deficits in patients with multiple sclerosis (MS), the most common primary demyelinating disease in humans. We used the Theiler's murine encephalomyelitis virus model of demyelination to investigate the role of major histocompatibility complex (MHC) class I and class II gene products in the development of functional and neurophysiological deficits following demyelination. We measured spontaneous clinical activity by two independent assays and recorded hind-limb motor-evoked potentials in infected class I-deficient and class II-deficient mice of an identical genetic background as well as in highly susceptible SJL/J mice. The results show that despite a similar distribution and extent of demyelinated lesions in all mice, only class I-deficient mice were functionally normal. We propose that the mechanism by which demyelinated class I-deficient mice maintain neurologic function results from increased sodium channel densities and the relative preservation of axons. These findings are the first to implicate a role for MHC class I in the development of neurological deficits following demyelination.

Fig. 1

Three-dimensional reconstruction of demyelinated lesions from chronically infected nonmutant H–2^b (β₂m^+/+), class I-deficient (β₂m^−/−), SJL/J and class II-deficient (Ab°) mice. The extent and distribution of demyelination is similar in β₂m^−/− (c and d), SJL/J (e and f) and class II-deficient (Ab°) (g and h) mice. β₂m^+/+ mice (a and b) show no demyelination. Green, gray matter; white, white matter; red, demyelinated lesions.

Fig. 2

Functional status of mice as measured by the Digiscan activity monitoring system, the activity wheel and conduction velocities in hind-limb motor-evoked potentials. Vertical activity was monitored hourly (over 3 days) for all groups of mice. a, The progressive decrease in nocturnal vertical activity from 0 to 180 days after infection is illustrated for susceptible SJL/J mice. b, A time course for SJL/J mice revealed a decrease in the ratio of nocturnal vertical activity as early as 60 days after infection when compared with nonmutant H–2^b (β₂m^+/+) mice. This activity was completely abolished by 270 days after infection. c, At 180 days after infection there was no difference observed between the mean nocturnal vertical activity ratios for infected nonmutant H–2^b (β₂m^+/+) and class I-deficient (α₂m^−/−) mice, indicating that hind-limb motor abnormalities were minimal in these mice. In contrast the ratios of mean nocturnal vertical activity for 180-day-infected SJL/J and 100-day-infected class II-deficient (Ab°) mice were significantly lower than those of nonmutant H–2^b (β₂m^+/+) and class I-deficient β₂m^−/− mice. d, Spontaneous activity measured by the activity wheel. The ratios of mean revolutions per day revealed the same pattern of normal activity in class I-deficient (β₂m^−/−) mice when compared with infected H–2^b (β₂m^+/+) mice, whereas infected SJL/J and class II-deficient (Ab°) mice displayed significantly decreased activities. e, Conduction velocities of 180-day-infected β₂m^−/− mice did not differ significantly from those of uninfected control β₂m^−/− mice or uninfected and 180-day-infected β₂m^+/+ mice. Conduction velocities for 180-day-infected SJL/J mice were slower when compared with uninfected SJL/J mice, 180-day-infected β₂m^+/+ mice and 180-day-infected β₂m^−/− mice. Conduction velocities for 100-day-infected, class II-deficient mice were also slower as compared with uninfected class II-deficient mice, 180-day-infected β₂m^+/+ and 180-day-infected β₂m^−/− mice. There were no differences observed between the mean conduction velocities for any of the uninfected controls. Uninfected, open symbols; chronically infected, shaded symbols. f, Representative tracings of recorded motor-evoked potentials from β₂m^+/+, β₂m^−/−, SJL/J and class II-deficient mice. Recordings from 180-day-infected β₂m^+/+ and β₂m^−/− mice are similar to their littermate uninfected controls. Recordings from 180-day-infected SJL/J and 100-day-infected class II-deficient mice showed prolonged latencies, dispersion of potentials and smaller amplitudes when compared with their uninfected controls. The arrow represents the point used to calculate latency to the beginning of the potential.

Fig. 3

Sodium channel distributions in uninfected (filled triangle) and chronically infected (open circle with dot) class I-deficient (β₂m^−/−) and SJL/J mice. a, A significant increase was observed in the sodium channel intensities of 323-day-infected β₂m^−/− mice when compared with their uninfected counterparts. b, In contrast, there was a significant decrease in sodium channel intensities of 270-day-infected SJL/J mice as compared with uninfected controls. a and b, A positive linear correlation (thickened gray lines, chronically infected groups; thin black lines, uninfected groups) exists between sodium channel and corresponding myelin proteolipid protein (PLP) intensities for uninfected SJL/J (r = 0.50), infected SJL/J (r = 0.20) and uninfected β₂m^−/− mice (r = 0.47). However, there is no positive linear correlation between sodium channel and PLP intensities for chronically infected β₂m^−/− mice (a), which can be explained by regions of high sodium channel intensities in areas of low PLP intensities. c, [³H]Saxitoxin autoradiography showed a 1.7-fold increase in the sodium channel grain density of 323-day-infected β₂m^−/− mice when compared with that of uninfected controls. In contrast, there was a 50% decrease in the sodium channel grain density of 270-day-infected SJL/J mice when compared with their uninfected controls. White bars, uninfected; black bars, chronically infected. Preincubation with 200-fold excess of unlabeled tetrodotoxin blocked approximately 50% of the [³H]saxitoxin labeling in an uninfected β₂m^+/+ mouse (data not shown). Representative examples of sodium channel staining (d, e and f) and PLP-myelin staining (g, h and I) in an uninfected β₂m^−/− mouse (d and g), a 323-day-infected β₂m^−/− mouse (e and h) and a 270-day-infected SJL/J mouse (f and i). Images for the sodium channel stain and their respective PLP stained counterparts were taken from the same field. A normal distribution of sodium channel (d) and myelin (g) is shown for an uninfected β₂m^−/− mouse. (Uninfected nonmutant mice had a comparable staining pattern.) Note the elevated and homogenous distribution of sodium channel staining (e) in an area of relative myelin loss (h) from an uninfected β₂m^−/− mouse. Note the severe loss of sodium channel intensity (f) in a similar area of low myelin (i) in a chronically infected SJL/J mouse. Even though the degree of myelin loss is similar in infected β₂m^−/− (h) and SJL/J (i) mice, there is more sodium channel expression in β₂m^−/− (e) compared with the SJL/J (f) mouse. Regions of negative staining were determined from the corresponding ultraviolet images to be cellular nuclei. Green, sodium channels; red, PLP.

Fig. 4

Representative examples of axons in the spinal cord white matter of chronically infected class I-deficient (β₂m^−/−) and SJL/J mice and uninfected class I-deficient and nonmutant H–2^b (β₂m^+/+) mice. Representative examples of antineurofilament (a-c) and Bielschowski (d-f) staining are shown for an uninfected β₂m^+/+ mouse (a), an uninfected β₂m^−/− mouse (d), a chronically infected (323 days postinfection) class I-deficient mouse (b and e) and a chronically infected (270 days) SJL/J mouse (c and f). A normal distribution of axons is shown for an uninfected β₂m^+/+ mouse (a) and an uninfected β₂m^−/− mouse (d). The staining pattern in uninfected animals of all groups is comparable. Axons are relatively well preserved in chronically infected β₂m^−/− mice despite the presence of inflammatory cells and demyelination. Both the neurofilament (b) and the Bielschowski (e) stains show that the axons in β₂m^−/− mice are neither significantly disrupted nor degenerating. In contrast the axons in chronically infected SJL/J mice are severely disrupted. The neurofilament stain (c) shows a significant loss of immunostainable material, whereas the Bielschowski stain (f) shows swelling axons (red arrows) which are actively undergoing degeneration.