Supplementary Fig. 1 and Movie 1. Increased dynamics of cortical microglia in EAE by
time lapse confocal imaging of brain slices. a) Diagram of methods used to stain brain slices
with Alexa-488 IB4. b) z-stack of live microglia in control animals showing few stained
processes, compared with chronic EAE 250 dpi. In the normal cortex, microglia have discrete
bodies and shortened processes that exhibit a weak staining with alexa 488-IB4. Notably, animals
with EAE 250 dpi exhibit an increased labeling with IB4 and an increased network of interacting
processes with highly dynamic branching (arrows) using the same parameters and concentration
of fluorochrome in naïve and EAE explants, as shown as well in the supplementary movie 1. For
each experiment, two independent areas in cortex were examined per animal (control n=2; EAE
n=3). Each movie is made of four loops at 10 fps in iMovie, where each frame is a maximumintensity projection from z-stacks of fluorescence images recorded every 5 min for a total of 1
hour (1.5 m axial spacing) with a 63 water immersion objective, 1.7 digital zoom, field of
view 360 m2.
File name: RasmussenMovS1 (Size: 4.3 Mb)
Supplementary Fig. 2. Increased apoptosis during EAE in non-neuronal cells. (a) The
number of TUNEL positive cells was quantified during acute disease and control and there was a
significant increase during acute EAE (EAE 68.338.4 versus control 2.671.3, p0.05). (b) The
number of activated Caspase-3 positive cells was also quantified and there was a significant
increase during acute disease (acute 118.06.5 versus control 7.001.5, p0.001, n=4
mice/group) compared to control but no difference when comparing the late relapse with the
control. (c) Shows TUNEL positive cell in close contact with a NeuN positive cell. (d) Example
of activated Caspase-3 positive cell in close contact with a neuron, (e) also shown in the
orthogonal-view (scale bar in c-e represents 5m).
Supplementary Fig. 3. MBP staining shows diffuse demyelination in corpus callosum in
EAE mice. (a-h) Immunohistochemistry for MBP show a persistent diffuse demyelination
throughout corpus callosum at all four time points in EAE mice with no decrease in MBP
staining for the age matched control groups (scale bar represents 200m). (i-l) Covariance of the
pixel intensity of the MBP staining over a distance of 300 m through corpus callosum show a
significant reduction in all four EAE groups compared to controls, [(i) acute 69.030.7 versus
control 101.21.1, p0.0001, (j) first remission 40.140.5 versus control 65.700.9, p0.0001,
(k) late relapse 54.180.8 versus control 78.630.9, p0.0001 and late remission 58.990.6
versus control 96.591.1, p0.0001, n=3 mice/group]. (m) The average MBP pixel intensity for
all four EAE groups combined was also significantly reduced compared to the average control
group (EAE 55.300.3 versus control 87.240.7, p0.0001).
Supplementary Fig. 4 and Movie 2. Increased dynamics of callosal microglia in EAE by
time lapse confocal imaging of brain slices. z-stack of live microglia in control animals
showing few stained process, compared with callosal microglia from animals with chronic EAE.
Normal microglia were stained in discrete numbers with short processes (arrows). In contrast,
during chronic EAE 250 dpi, there was a persistent activation of microglia with strong staining
for IB4 and an increased numbers of cell bodies (red arrows) with increased surveillance and
process motility (blue arrows), as shown in the supplementary movie 2. For each experiment, the
live imaging was repeated in two independent areas of the corpus callosum in independent EAE
mice with age-matched controls (control n=2; EAE n=3). All the parameters and the scales were
maintained in controls and EAE samples. Each movie is made of four loops at 10 fps in iMovie,
where each frame is a maximum-intensity projection from z-stacks of fluorescence images
recorded every 5 min for a total of 1 hour (1.5 m axial spacing) with a 63 water immersion
objective, 1.7 digital zoom, field of view 360 m2.
File name: RasmussenMovS2 (Size: 4.6 Mb)