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. 2017 Oct 12;8:703. doi: 10.3389/fphar.2017.00703

FIGURE 4.

FIGURE 4

Remyelinating potential of transplanted meningeal-derived oligodendrocytes. (A–C) Brightfield images of spinal cord transversal sections of healthy, LPC-control, and LPC-transplanted rats stained with specific myelin staining LFB. LFB allows the identification of the myelin content of the tissue (blue) from the demyelinated area (white). While spinal cord sections of healthy rat did not show any evidence of demyelination (A), spinal cord sections of LPC-control rats showed a focal demyelination in the injection sites, as indicated by the blue arrow (B). (C) Spinal cord sections of LPC-transplanted rats showed an higher intensity of the LFB staining around the injection site compared to LPC-control rat sections. (D) The graph represents the percentage of the myelin content in the dorsal column of the spinal cord sections of healthy (38.94% ± 1.2%), LPC-control (26.42% ± 0.6%), and LPC-transplanted (33.97% ± 1.1%) rats, calculated as myelin positive pixels in the dorsal column among the pixels of the total area of the dorsal column. The blue dashed lines in (A–C) indicate the dorsal column areas of the spinal cord sections of healthy, LPC-control, and LPC-transplanted rats, that represent the LPC-lesioned area considered for the myelin content quantification. The analysis shows that transplantation of meningeal-derived oligodendrocytes resulted in a significantly increased myelin content percentage in LPC-transplanted sections compared to LPC-controls sections. Quantitative data are expressed as means ± SEM; n = 3 in healthy animals, n = 6 in LPC animals; ∗p ≤ 0.05, ∗∗∗∗p ≤ 0.0001. The average value was calculated for healthy control, LPC-control and LPC-transplanted rats (n ≥ 20 slices/animal). (E,F) Immunostaining for GFP (green) and TO-PRO3 nuclei (blue) in a spinal cord section of a LPC-transplanted rat at 1 DPT, showing that eGFP+ meningeal-derived oligodendrocytes were localized inside the spinal cord parenchyma close to the LPC lesion site. The white dashed line in (E) indicates the dorsal columns of the spinal cord, (F) is a higher magnification of the box in (E). (G-I) Immunostaining of a spinal cord section of a LPC-transplanted rat at 21 DPT, showing that eGFP+ meningeal-derived oligodendrocytes (green) co-express the specific marker for mature oligodendrocytes, MBP (red). Merged image in (G); GFP (green) and TO-PRO3 (blue) in (H); MBP (red) and TO-PRO3 (blue) in (I). (J–L) Immunostaining of a spinal cord section of a LPC-transplanted rat at 21 DPT, showing that eGFP+ meningeal-derived oligodendrocytes (green) are in close contact to the neuron neurofilament, stained with NF160 (white arrows in (J–L). Merged image in (J); GFP (green) and TO-PRO3 (blue) in (K); NF160 (red) and TO-PRO3 (blue) in (L). (E) and (F) are maximum Z-projection images of confocal images. Scale bars: 1 mm (A–C), 500 μm (E), 20 μm (F,H,I,K,L), 40 μm (G,J).