Analysis of nociception, sex and peripheral nerve innervation in the TMEV animal model of multiple sclerosis

Abstract

Although pain was previously not considered an important element of multiple sclerosis (MS), recent evidence indicates that over 50% of MS patients suffer from chronic pain. In the present study, we utilized the Theiler's murine encephalomyelitis virus (TMEV) model of MS to examine whether changes in nociception occur during disease progression and to investigate whether sex influences the development of nociception or disease-associated neurological symptoms. Using the rotarod assay, TMEV infected male mice displayed increased neurological deficits when compared to TMEV infected female mice, which mimics what is observed in human MS. While both male and female TMEV infected mice exhibited thermal hyperalgesia and mechanical allodynia, female mice developed mechanical allodynia at a faster rate and displayed significantly more mechanical allodynia than male mice. Since neuropathic symptoms have been described in MS patients, we quantified sensory nerve fibers in the epidermis of TMEV-infected and non-infected mice to determine if there were alterations in epidermal nerve density. There was a significantly higher density of PGP9.5 and CGRP-immunoreactive axons in the epidermis of TMEV-infected mice versus controls. Collectively these results indicate that the TMEV model is well suited to study the mechanisms of MS-induced nociception and suggest that alterations in peripheral nerve innervation may contribute to MS pain.

Figure 1. Assessment of motor coordination and balance (Rotarod test) in TMEV-infected and uninfected male and female mice

An accelerating Rotamex Rotorod (Columbus Instruments, Columbus OH) was used to test the effects of TMEV-induced demyelination on motor coordination. Mice were tested on the rotarod at an accelerating speed between 5 and 40 rpm over a 20 minute test period and the run time was recorded in seconds. TMEV infected mice (n=20) had significantly shorter run times when compared to uninfected controls (n=15; *p<0.0001). TMEV infected-female mice performed at a significantly higher level than TMEV-infected male mice (†p≤0.0025). Control male and female mice were not significantly different from each other (p=0.921).

Figure 2. Development of mechanical allodynia in TMEV-infected male and female mice

Graphs illustrating the mean paw withdrawal score (paw withdrawal frequency as a percent) for each mouse (individual dots) at various time points. The results for TMEV-infected mice are shown in the top two plots, while the data for the non-infected controls are shown in the bottom two plots. The straight lines in each plot represent fit of a mixed model assuming linear time effects. The intercept is the same for each sex (t=−1.58, df=135, p=.11). The slope is equal to zero for both male and female controls (overall test, t=0.66, df=469, p=.50). In the TMEV condition, separate, nonzero slopes were required for males and females. For females, the estimated slope was .0225 units per day (for testing the slope equal to zero, t=17.21, df=469, p<.0001), while for males the slope was estimated to be 0.0163 (for testing the equality of slopes for males and females, t=−4.01, df=469, p=.0001). The data indicate that TMEV-infected mice developed significant mechanical allodynia over time compared to control uninfected mice and that TMEV-infected female mice develop mechanical allodynia at a faster rate than male TMEV-infected mice.

Figure 3. Development of thermal hyperalgesia in TMEV-infected male and female mice

Graphs illustrating the mean tail withdrawal latency for each mouse (individual dots) at various time points. The results for TMEV-infected mice are shown in the top two plots, while the data for the non-infected controls are shown in the bottom two plots. The straight lines in each plot represent the fit of a mixed model assuming linear time effects. We used the same modeling strategy that we utilized for evaluating the mechanical-induced paw withdrawal scores illustrated in figure 1. We found that TMEV-infected mice developed significant thermal hyperalgesia over time compared to control uninfected mice. Similar to what we observed with mechanical von Frey testing, we found that thermal latencies did not vary over time in the control condition (t=−1.35, df=510, p=.18). The slope in the TMEV condition is significantly different from zero (t=−12.72, df=510, p <.0001) indicating that TMEV mice develop thermal hyperalgesia over time.

Figure 4. Graphs demonstrating a lack of correlation between nociception (thermal hyperalgesia and mechanical allodynia) and motor deficits (rotarod)

(A) There was a lack of correlation between thermal hyperalgesia as measured by tail withdrawal latency (y axis) and rotarod mechanical deficits in TMEV infected mice at day 180 PI. (B) There was also a lack of correlation between mechanical allodynia as determined with von Frey filaments and rotarod at day 180 PI.

Figure 5. Photomicrographs of Intra-epidermal PGP9.5 and CGRP Immunoreactivity

Representative photomicrographs at low power magnification (100Xmag, upper panels) and high power magnification (1200Xmag, four lower panels) of dorsal skin from SJL/J mice, infected and non-infected. Skin was collected at day 180 post-infection and double-immunostained with anti-PGP9.5 and anti-CGRP. (a) Control, non-infected mouse epidermis and dermis exhibit lower levels of PGP9.5-immunoreactivity as compared to (b) TMEV-infected mice. At higher magnification (c and e) control, non-infected mouse epidermis exhibits lower levels of PGP9.5- and CGRP-immunoreactivity as compared to (d and f) TMEV-infected mice. (Arrows designate the co-localization of CGRP and PGP9.5-immunoreactive nerve fibers in the epidermis both in the control, c and e, and in the TMEV-infected mice, d and f.) Interestingly CGRP-immunoreactivity was enhanced in epidermal keratinocytes in the TMEV-infected skin samples (f) compared to controls (e). Scale bar for lower magnification (Olympus UPlanFl 10X/0.30) image is 500 microns and for higher magnification (Olympus UPlanApo_60X/1.4 oil) scale bar is 10 microns.

Figure 6. Quantification of Intra-epidermal Nerve Density

The immunoreactivity density counts for PGP9.5 and CGRP were calculated by determining immunopositive pixel density area per total intra-epidermal pixel area at 120Xmag. Data are presented as mean and SEM, analyzed using ANOVA and Fisher’s post-hoc test (*p<.05).