Recovery from chronic demyelination by thyroid hormone therapy: myelinogenesis induction and assessment by diffusion tensor magnetic resonance imaging

Abstract

The failure of the remyelination processes in multiple sclerosis contributes to the formation of chronic demyelinated plaques that lead to severe neurological deficits. Long-term cuprizone treatment of C57BL/6 mice resulted in pronounced white matter pathology characterized by oligodendrocyte depletion, irreversible demyelination and persistent functional deficits after cuprizone withdrawal. The use of a combination of in vivo diffusion tensor magnetic resonance imaging (DT-MRI) and histological analyses allowed for an accurate longitudinal assessment of demyelination. Injection of triiodothyronine (T(3)) hormone over a 3 week interval after cuprizone withdrawal progressively restored the normal DT-MRI phenotype accompanied by an improvement of clinical signs and remyelination. The effects of T(3) were not restricted to the later stages of remyelination but increased the expression of sonic hedgehog and the numbers of Olig2(+) and PSA-NCAM(+) precursors and proliferative cells. Our findings establish a role for T(3) as an inducer of oligodendrocyte progenitor cells in adult mouse brain following chronic demyelination.

Figures

Figure 1.

Experimental groups and summary of DT-MRI and histological examinations. A, The longitudinal examination was conducted starting at w0 time point (week before any treatment) and continuing at w12 (week 12 of cuprizone treatment), w12 + 3, w12 + 6, and w12 + 12 (at 3, 6, and 12 weeks after cuprizone removal). B, Results of rotarod test, performed twice a week in each experimental group. The mice were considered as unaffected when performing minimum 2 min (120 s) on the rotarod. The figure shows normal scores for the mice of control group, while the mice subjected to cuprizone diet show clinical signs from the fourth week of regimen, with a relapsing remitting pattern. The mice receiving T3 injections after cuprizone withdrawal (Cuprizone +T3 group) improve considerably their clinical score during the next 12 weeks of observation. On the contrary, only weak improvements were quantified in this period for the mice allowed to recover spontaneously, without any stimulation (Cuprizone group).

Figure 2.

A, B, Representative sagittal brain T2-weighted images of individuals from groups 1 (A) and 2 (B) examined at different time points. At w0, corresponding to the exam prior to the cuprizone ingestion, the myelinated areas, corpus callosum (arrows) and cerebellum showed hypointense signals. Enlarged ventricles corresponding to the hyperintense signal are evident after 12 weeks of cuprizone diet in both groups (w12). No significant changes of the T2 images pattern are observed during the w12 to w12 + 12 period in mice from group 1 (A). Partial regain in the hypointensities of corpus callosum and cerebellum are noticed in the group receiving T3 injections (group 2, B) during w12 to w12 + 12 time interval, with spectacular decrease in the ventricle volumes. The T2-weighted images pattern in control mice is identical with that observed at w0 time point in groups 1 and 2 and is not presented. C, Investigated ROIs overlaid on a sagittal DWI of a normal mouse brain at w0.

Figure 3.

A, B, Fractional anisotropy maps of representative individuals from experimental groups 1 (A) and 2 (B), followed longitudinally at different time points. Normal brain images at w0 show high anisotropic values in the well formed corpus callosum (white arrows). FA maps resulted from DT-MRI exams at w12 display very low values of this parameter all along the corpus callosum. No significant recovery of the FA is visible during the 12 weeks after the cuprizone removal in the brain images of mouse belonging to group 1 (A). Gradual increase in the FA is observed in the brain white matter of the mouse receiving T3 treatment after returning to normal diet (B, w12 + 3; w12 + 6; w12 + 12). The FA pattern in control mice is identical with that observed at w0 time point in group 1 and 2 and is not presented. FA values are distributed on a scale from 0 (blue) to 1 (red).

Figure 4.

Representative images of double immunostaining (merged) of the mice corpora callosa with anti-MBP and anti-CA II antibodies. The anti-MBP antibody detects the myelin in red, while oligodendrocytes are green for anti-CA II antibody. Higher magnification of regions of corpus callosum is presented in the bottom of each image. A, At w0, before the cuprizone treatment, many green CA II+ oligodendrocytes (arrows) and MBP+ myelinated fibers were detected. The same staining patterns were obtained in the sections of control mice at each time point and are omitted from presentation. B, After 12 weeks of cuprizone insult (w12), very few oligodendrocytes and only scattered myelinated fibers were noticed. C–E, Failure of consistent remyelination is quantified after the cuprizone withdrawal in mice belonging to group 1 at w12 + 3 (C), w12 + 6 (D), w12 + 12 (E). F–H, Important progressive recovery of the myelin (MBP red staining) and repopulation by a great number of oligodendrocytes (CA II green staining) are evident in the corpus callosum of mice from group 2, at w12 + 3 (F), w12 + 6 (G), and w12 + 12 (H). Scale bar, 200 μm.

Figure 5.

Representative electron micrographs of the mouse corpus callosum. A, At w0, before cuprizone treatment, well myelinated axons are observed. Similar electron micrographs were obtained from sections of control mice at each time point (data not shown). B, After 12 weeks of cuprizone insult (w12), most axons are demyelinated and have small caliber. Scale bar, 1 μm. C–E, Failure of consistent remyelination is quantified after cuprizone withdrawal in mice belonging to group 1 at w12 + 3 (C), w12 + 6 (D), w12 + 12 (E). F–H, Important progressive recovery in both, number of myelinated axons and myelin thickness is evident in the corpora callosa of mice from group 2 (that received T3 hormone), at w12 + 3 (F), w12 + 6 (G), and w12 + 12 (H). I, Number of myelinated axons/100 μm2 counted in semithin sections of the corpus callosum in animals from groups 1 and 2, at each experimental time point. J, Axonal caliber measurements performed on electron micrographs acquired from the corpus callosum region of mice from groups 1 and 2, at each experimental time point. * represents the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) versus the control group, while # expresses the statistically significant differences (#p < 0.05, ##p < 0.001) among groups 1 (cuprizone treatment) and 2 (cuprizone plus T3 treatments).

Figure 6.

A–F, Time course of D⊥ (A, C, E) and D‖ (B, D, F) in the genu (A, B) and splenium (C, D) of corpus callosum and cerebellum (E, F) in all experimental groups. * represents the statistical significance (*p < 0.05, **p < 0.001) versus the control group, while # expresses the statistically significant differences (#p < 0.05, ##p < 0.001) among groups 1 (cuprizone treatment) and 2 (cuprizone +T3 treatments).

Figure 7.

A–C, Brain section immunolabeling with anti-CD45 (A), anti-PSA-NCAM (B), and anti-NG2 (C) obtained at w12 + 3 in the corpus callosum of animals from groups 1, 2, and 3. We chose to present the data at w12 + 3 because they are the most representative for the comparison between mice receiving T3 treatment and remyelinating (group 2) and chronic demyelinated mice (group 1). Magnification ×400.

Figure 8.

A–C, Double immunostaining (CA II and PCNA) of the corpus callosum of mice from group 1 (A), group 2 (B), and group 3 (C). Numerous cells double stained with PCNA and CA II markers were present in the corpus callosum of T3-treated mice (B). The double-labeled cells were absent from the corpus callosum of chronically demyelinated mice (A) or control mice (C). Magnification ×600.

Figure 9.

A–H, Olig2 (A–C), Shh (D–F), and MBP (G–I) immunoperoxidase staining in splenium of corpus callosum. In control mice (A) a significant number of Olig2+ cells are present while the number was dramatically reduced after cuprizone treatment (B). A massive reappearance of Olig2+ (C) and Shh+ cells (F) was observed under the influence of T3 treatment (C, F). Shh+ cells were almost absent in control mice (D) and a weak staining of limited number of Shh+ cells were observed in cuprizone-treated mice (E). The induction of Olig2+ and Shh+ cells was accompanied by a massive recovery in MBP immunostaining (I) after a dramatic reduction of MBP immunoreactivity in cuprizone-treated mice (H) compared with the control (G). Magnification ×250. The number of Olig2 cells in splenium of corpus callosum (J) was counted in 4–6 fields per tissue section photographed under microscope using an objective ×10 and two sections per animal. The number of CA II+ cells/10,000 μm2 counted in the corpus callosum (K). The CA II+ cells were counted using the Image J software on micrographs taken with the ×40 objective along the entire corpus callosum. Minimum of 5 slides/animal were analyzed (n = 5/group). * represents the statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001) versus the control group, while # expresses the statistically significant differences (###p < 0.001) among groups 1 (cuprizone treatment) and 2 (cuprizone +T3 treatments).

Figure 10.

A, B, Immunofluorescence of NG2 revealed with Alexa Fluor 546-conjugated anti-rabbit IgG in cuprizone-treated mice (A) and cuprizone+T3-treated mice (B) at w12 + 3. C, D, Double labeling of Olig2 immunoperoxidase detected with VIP in cell nuclei (dark color) and CA II immunofluorescence in corpus callosum of cuprizone+T3-treated mouse (C, D). CA II+ cells and Olig2+ cell nuclei (arrows) are predominantly present in demyelinated area. Magnification: A–C, ×400; D, ×900.