Chronic demyelination-induced seizures

Abstract

Multiple sclerosis (MS) patients are three to six times more likely to develop epilepsy compared to the rest of the population. Seizures are more common in patients with early onset or progressive forms of the disease and prognosticate rapid progression to disability and death. Gray matter atrophy, hippocampal lesions, interneuron loss, and elevated juxtacortical lesion burden have been identified in MS patients with seizures; however, translational studies aimed at elucidating the pathophysiological processes underlying MS epileptogenesis are limited. Here, we report that cuprizone-mediated chronically demyelinated (9-12weeks) mice exhibit marked changes to dorsal hippocampal electroencephalography (EEG) and evidence of overt seizure activity. Immunohistochemical (IHC) analyses within the hippocampal CA1 region revealed extensive demyelination, loss of parvalbumin (PV⁺) interneurons, widespread gliosis, and changes in aquaporin-4 (AQP4) expression. Our results suggest that chronically demyelinated mice are a valuable model with which we may begin to understand the mechanisms underlying demyelination-induced seizures.

Keywords: axon damage; cuprizone; electroencephalography; hippocampus; multiple sclerosis; parvalbumin.

Figure 1

Hippocampal electroencephalogram (EEG) changes occur in chronically demyelinated mice. A) Representative images of a control mouse fed normal diet (normal), an experimental mouse that was fed cuprizone (CPZ) diet for nine weeks (9 wk CPZ) undergoing seizure-like behavior during handling (seizure), and an image of a normal mouse with an intrahippocampal probe tethered to an external EEG recording setup to allow freedom of movement (EEG setup). B) Nearly 30% of mice on CPZ diet for 8 weeks presented overt seizure during handling. Seizure activity increased to 55% in 9 wk CPZ mice and 65% in 12 wk CPZ mice (total of 20-45 mice observed over 3 different experiments). C) Representative 30 min recordings from a normal, 9 wk CPZ, & 12 wk CPZ animal are shown. D) Recording period highlighted in blue box from A was magnified to better assess changes in activity. E) Frequency analysis was performed on 4 hours of EEG recordings from the same period in normal [n=9], 9 wk CPZ [n=7], and 12 wk CPZ [n=10] groups. No significant changes were observed in any frequency between normal and 9 wk CPZ groups. Similarly, 12 wk CPZ mice showed power equivalent to normal across all frequencies, but exhibited increased theta, alpha, and beta frequency power compared to 9 wk CPZ (theta *p≤0.05, η2=0.2928; alpha *p≤0.05 η2=0.2571; beta *p≤0.05 η2=0.3203, one-way ANOVA with Tukey's posttest for multiple comparisons.

Figure 2. Demyelination in the corpus callosum (CC) and hippocampus (hipp) during chronic cuprizone diet

A) Representative 10× montages of coronal brain section immunostained for myelin basic protein (MBP-green) and double-stranded DNA (DAPI-red) from normal, 9 wk CPZ and 12 wk CPZ mice show decreased MBP staining in the CC and hippocampi of 9 wk CPZ and 12 wk CPZ group. Scale bar: 1 mm, * indicates injury at the site of EEG probe implant. B) Representative 10× magnified image of CC, scale bar 100 μm (i) and 40× magnified image of hippocampus area showing the stratum radiatum (SR) and stratum lacunosum-moleculare (SLM) (ii) immunostained for MBP (green) and DAPI (red) antibodies. A decrease in myelinated fibers was observed in 9 wk and 12 wk CPZ panels. 40× magnified images of the CC (iii) and CA1 (iv) immunostained for olig2 (green) and DAPI (red) were analyzed for olig2⁺ oligodendrocyte (OL) lineage cell numbers. Scale bar in ii, iii, and iv is 10 μm. C) Quantification of MBP staining intensity in the CC normalized to area measured showed a significant decrease in 9 wk CPZ (**p≤0.01) and 12 wk CPZ groups (**p≤0.01) compared to normal mice. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.4373. D) No difference was identified in the number of olig2⁺ nuclei counted in the CC of normal, 9 wk CPZ, and 12 wk CPZ mice. 7-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons. E) Fewer post-mitotic olig2⁺CC-1⁺ OLs were counted in the CC of 9 wk CPZ (****p<0.0001) and 12 wk CPZ (***p<0.0001) groups as compared to normal mice. 7-10 animals/group, oneway ANOVA with Tukey's posttest for multiple comparisons, η²=0.8933. F) Apoptotic olig2⁺caspase 3⁺ cells accounted for 44.29±2.879% of all olig2⁺ OL lineage cells in the CC of 9 wk CPZ mice (9 wk CPZ vs normal ****p<0.0001) and 46.39±1.661% of total olig2⁺ OL lineage cells in the CC of 12 wk CPZ mice (12 wk CPZ vs normal ****p<0.0001). No difference was observed between 9 wk CPZ and 12 wk CPZ groups. 7-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.9784. G) MBP immunostained areas of CA1 SR/SLM subregions were significantly reduced in 9 wk CPZ (****p<0.0001) and 12 wk CPZ groups (****p<0.0001) compared to normal mice. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.7453. H) No significant change was observed in olig2+ OLs between normal and 9wk CPZ mice, but a 20% decrease of olig2⁺ OLs was observed in 12 wk CPZ compared to normal (*p≤0.05). 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.2725. I) Post-mitotic olig2⁺CC-1⁺ OLs counted in the CA1 were significantly decreased in 9 wk CPZ mice (**p≤0.01) and 12 wk CPZ mice (****p<0.0001) compared to normal. 9-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.5471. J) Cleaved caspase 3 labeled olig2⁺ OL lineage cells were not observed in any groups analyzed. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons.

Figure 3. Macrophage/microglia infiltration, reactive astrocytes, and altered AQP4 expression are observed in the CA1 of chronically cuprizone-fed mice with seizures

A) Representative 40× magnification images of Iba1 (red) and DAPI (blue) immunostained CA1 SO/SR from normal, 9 wk CPZ, and 12 wk CPZ mouse coronal brain sections showing increased Iba1 reactivity in 12 wk CPZ mice. Scale bar: 10 μm. B) Representative 40× magnification images of GFAP (green), AQP4 (red) immunostaining, and DAPI (blue) stained CA1 SO/SR of normal, 9 wk CPZ, and 12 wk CPZ mice. The number of GFAP⁺ astrocytes and staining intensity per cell increased in the CA1 of 9 wk and 12 wk CPZ mice and displayed hypertrophic morphology characteristic of reactive astrocytes, membrane varicosities, and loss of non-overlapping process domains (arrowheads). In normal mice, AQP4⁺ staining followed vasculature and co-localized with GFAP at the site of astrocyte end foot processes contacting blood vessels. AQP4 staining decreased in 9 wk CPZ mice, but GFAP staining increased proximal to vasculature. AQP4 reactive area returned to normal levels in 12 wk CPZ mice and GFAP⁺ reactive astrocytes did not accumulate near blood vessels. Scale bar: 10 μm. C) Iba1⁺ immunostaining in the CA1 was quantified as a percent of imaged field in normal, 9 wk CPZ, and 12 wk CPZ mice. Iba1⁺ microglia/macrophages occupied approximately 10—13% of the imaged CA1 in normal and 9 wk CPZ mice, but increased more than three-fold in 12 wk CPZ mice to 47.29±10.98% (12 wk CPZ vs normal, **p≤0.01; 12 wk CPZ vs 9 wk CPZ **p≤0.01). 7-9 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.5128. D—E) Astrocytes were quantified by counting number of GFAP⁺ cells/area. A significant increase was observed in the CA1 of 9 wk CPZ (***p≤0.001) and 12 wk CPZ (***p≤0.001) groups compared to normal mice. GFAP staining intensity (represented in arbitrary units “AU”) per astrocyte also rose significantly in 12 wk CPZ mice compared to normal mice *p≤0.05), indicating increased GFAP expression per cell. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, GFAP⁺ cells/mm² η²=0.5658; GFAP intensity per cell η²=0.2514. F) AQP4 reactive area was significantly decreased in the CA1 of 9 wk CPZ mice compared to normal (**p≤0.01) and 12 wk CPZ groups (****p<0.0001). 7-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.5668.

Figure 4. CC axon pathology, reduced CA1 pyramidal cell layer thickness and loss of PV immunostaining in the CA1 of chronically demyelinated mice with seizures

A—B) Representative 40× magnification images from normal, 9 wk CPZ, and 12 wk CPZ coronal brain sections showing the CC and the SR/SLM regions of the CA1. The coherent linear morphology of Thy1-YFP⁺ axons in the normal CC was lost with chronic demyelination. Thy1-YFP⁺ axon varicosities (arrowheads), indicative of transected axons or impaired protein transport, were observed in 9 wk and 12 wk CPZ groups. No axon or dendrite swelling/blebbing was observed in SR or SLM. Scale bar: 10 μm. C—D) Representative 40× magnification images of CA1 pyramidal layer immunostained for NeuN+ (green), DAPI, (blue), and parvalbumin (PV; red) from normal, 9wk CPZ and 12 wk CPZ groups. A decrease in overall CA1 pyramidal neuron layer thickness and loss of PV⁺ neurons was observed in 9 wk CPZ and 12 wk CPZ CA1 pyramidal layer. Scale bars: 10 μm. E) Quantification of axon blebs in the CC showed a significant increase in both 9 and 12 wk CPZ groups compared to the normal group (9 wk CPZ vs normal ****p<0.0001; 12 wk CPZ vs normal *p≤0.05). Blebbing in the CC of 9 wk CPZ mice was significantly higher than 12 wk CPZ mice (****p<0.0001), reflecting fewer Thy1-YFP callosal axons. 8-10 animals/group, oneway ANOVA with Tukey's posttest for multiple comparisons, η²=0.9258. F—G) Quantification of pyramidal cell layer thickness was determined by measuring the total height of NeuN⁺ nuclei in the pyramidal cell layer. Compared to normal, a significant decrease was observed in pyramidal cell layer thickness of 12 wk CPZ mice (*p≤0.05). However, total pyramidal layer NeuN+ neurons were not statistically different between groups. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons; pyramidal layer height η²=0.2458. H) Quantification of PV⁺ interneuron soma in the CA1 region pyramidal cell layer showed a significant decrease in only 12 wk CPZ mice *p≤0.05) as compared to normal group. 8-10 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons, η²=0.4372.

Figure 5. Status of Ki-67⁺ doublecortin⁺ cell population in the CA1 of chronically demyelinated mice with seizures

A) Representative 40× magnified images of CA1 pyramidal cell layer immunostained for doublecortin (DCX) and Ki-67 in normal, 9 wk CPZ, and 12 wk CPZ mice. DCX⁺ cells with stellate morphology resembling astrocytes or microglia were observed in the CA1 SO and overlying white matter tract of 9 wk CPZ and 12 wk CPZ mice. Ki-67⁺ nuclei were rarely detected in the CA1 of any group, and did not co-localize with DCX. Scale bar: 10 μm. B) DCX⁺ cells were significantly increased in the CA1 SO of 9 wk CPZ (****p<0.0001) and 12 wk CPZ mice mice (**p≤0.01). 8-9 animals/group, one-way ANOVA with Tukey's post test for multiple comparisons, η2=0.6077. C) Ki-67⁺ nuclei counted were not significantly different between groups. 8-9 animals/group, one-way ANOVA with Tukey's posttest for multiple comparisons