Long-term analysis of cuprizone-induced de- and remyelination

We used the toxin cuprizone to study the long-term effect of demyelination on axonal preservation and motor functional recovery. Feeding mice with 0.2-0.3% cuprizone (Sigma-Aldrich) for 5-6 weeks results in massive demyelination of white matter tracts in the brain, mainly the corpus callosum. Removing cuprizone from the diet for more than 3 weeks results in extensive remyelination. To observe the difference in the effect of a single or repeated demyelinating events, we submitted a group of male mice for 2 cycles of 5 weeks of 0.25% cuprizone, allowing 4 weeks of recovery in between (named “DD” or “double demyelination” group). In addition, another group of age- and sex-matched mice (named

“SD” or “single demyelination” group) were submitted to a single demyelinating event by feeding them with cuprizone for 5 weeks (simultaneous with the second cycle of cuprizone in the DD group). A control group received normal milled chow. For a scheme of the experimental design, see Figure 3.10.

Figure 3.10: Experimental design of cuprizone treatment. 8-week-old mice were fed cuprizone for 5 weeks, were then allowed to recover for 4 weeks and finally received cuprizone for 5 more weeks (double demyelination, or DD). A second group of mice (single demyelination, or SD) were fed cuprizone for 5 weeks simultaneous to the second demyelination of the DD group as indicated. Animals were switched to a normal diet for 28 weeks until the end of the experiment. Age-matched controls were fed with a normal diet throughout the experiment. Motor Skill Sequence analysis (MOSS) was performed at the times indicated.

Animals were first habituated to training wheels composed of regularly spaced crossbars for two weeks.

Subsequently, running performance on complex wheels was recorded for an additional week.

The effectivity of the demyelinating paradigm using cuprizone was evaluated by staining coronal sections with Luxol Fast Blue - Periodic Acid Schiff (LFB-PAS) staining and as-sessing myelination level with a scale ranging from 0 (no demyelination) to 3 (complete de-myelination). We confirmed that demyelination occurred after each cuprizone treatment, and extensive remyelination was observed 28 weeks after the cuprizone was removed from the diet. As expected, the control group, which received normal milled chow, presented no demyelination (Figure 3.11).

Figure 3.11: Cuprizone-induced demyelination and remyelination.(A) Representative LFB/PAS-stained corpus callosum of untreated control (left) and after 5 weeks of cuprizone treatment (right). Extensive callosal demyelination is evident in the right panel. Scale bar: 200µm. (B) Semi-quantitative analysis of callosal demyelination (n=3-9 animals per group) and compared to age-matched control (n=2-6). A score of 3 corresponds to maximal demyelination, a score of 0 represents no detectable demyelination. For the first demyelination (left), at-test was applied. For second demyelination (center) and final time point (right), if ANOVA indicated significant differences in the main effect (p<0.05), Tukey test pair-wise comparison was applied. ∗∗p<0.01. Data shown as mean + SEM. DD: Double demyelination, SD: Single demyelination.

First demyelination: week 5, second demyelination: week 14, final time point: week 42 (see also Fig. 1a).

3.2.2 Late motor decline after accomplished remyelination

To understand whether demyelination can have long-term consequences on the function of the affected axons despite remyelination, we used a motor skills test called Motor Skill Se-quence (MOSS). It has been shown that MOSS is useful in assessing behavioral outcome as an indication of the changes in the functional state of the corpus callosum (Schalomon and Wahlsten, 2002; Liebetanz and Merkler, 2006). In this test, mice are allowed to voluntarily run in “complex” wheels with irregularly spaced crossbars following a 2-week training pe-riod in regular wheels in individual cages. Since the animals need to constantly adapt the step length in the complex wheels, this movement requires a bi-hemispherical coordination that involves the corpus callosum, as it is the largest white matter tract connecting both cortical hemispheres. The time, velocity and running events are recorded automatically 24 hours a day during seven days. The number of runs, time spent running and accumulated distance are considered to reflect the animal’s motivation and fitness, whereas the max-imum velocity (Vmax) and maxmax-imum distance (Dmax) accomplished during a running bout reflect the highest performance in bi-hemispherical motor coordination.

We assessed the running performance of the mice on three occasions during recovery: 6 weeks, 20 weeks and 28 weeks after cuprizone was removed from the diet (seeFigure3.10).

At the first time point of MOSS analysis (6 weeks after cuprizone removal), the maximal velocity and maximal accumulated distance in one run were reduced in treated animals compared to controls (Figure 3.12, left panel). This confirms previous findings that showed reduced maximal coordination capacity after 6 weeks of recovery in the cuprizone model, measured by MOSS (Liebetanz and Merkler, 2006). No differences were observed between the SD and the DD groups. At 20 weeks of recovery, there was no significant difference in any of the parameters measured by MOSS between the treated and control animals (Figure 3.12, central panel). This may indicate that extended remyelination or compensation mechanisms through cortical plasticity contribute to a functional recov-ery after cuprizone treatment. Strikingly, in the final time point of MOSS evaluation, 28 weeks after recovery, animals treated with cuprizone once again exhibited a decreased per-formance in the Vmax and Dmax measurements (Figure3.12, right panel). No difference was detected between the SD and DD groups. This indicates that, regardless of the num-ber of demyelinating events, in the long-term cuprizone treated animals entered a phase of motor decline, revealing latent functional deficits as a consequence of oligodendrocyte death and myelin loss.

Figure 3.12:Late-onset latent motor deficits as measured by wheel running performance af-ter remyelination.Animals received one (shown in red) and two (shown in green) cycles of cuprizone to induce demyelination and were allowed to recover for 28 weeks during which MOSS performance was com-pared to age- and sex-matched controls (shown in black). Recording was preceded by a two-week training session (day -1: performance of last day on training wheels, for details see material and methods). Complex wheel performance (day 1 to 7) is shown as follows: 6 weeks (left panels), 20 weeks (central panels) and 28 weeks (right panels) after cuprizone withdrawal. Differences between the cuprizone and control group with regard to wheel running performance were calculated by repeated measurements ANOVA. If ANOVA indi-cated significant differences in the main effect (p<0.05), Fisher’s LSD post-hoc tests were applied.∗p<0.05,

∗∗p<0.01, rpm: revolutions per minute (n=8-12 animals per group). DD: Double demyelination, SD: Single demyelination. Distac: distance in meters accumulated in 24 hours; Dmax: maximum distance per run; Nrun, number of individual runs in 24 hours; Vmax: maximum running velocity in revolutions per minute in 24 hours.

3.2.3 Cortical thickness and neuronal preservation after cuprizone