Quantification of myelin on mouse cerebellar slice preparation

Quantification of spontaneous remyelination (control) was compared to remyelination in the presence of BTKi at DIV13. Immunostainings were performed for PLP (myelin), Caspr (paranodes) and Calbindin (neuron) at DIV13 in order to quantify the number of myelinated segments and intact paranodal structures. Images were acquired using a Zeiss Axio-Imager-Apotome. Series of Z-sections were performed at 0.5 μm increment. Maximum orthogonal projection of images was carried out using Fiji software (NIH, Bethesda, Maryland). To analyze PLP expression (Fig. 1), images were quantified for labeled area fraction (expressed in %) by automated counting using Image J software. Briefly, images were normalized by subtracting background and an automatic thresholding (MaxEntropy) was applied. Mean values of area fraction (expressed in %) were obtained from images (n = 27) of cerebellum slices treated with LPC (n = 13) vs control (n = 14).

Increased BTK signal upon demyelination. Cerebellar organotypic slices from P9 Plp-GFP mouse (green oligodendrocytes and myelin) were maintained in culture (A) or subjected to LPC treatment at 6 days in vitro (DIV) for 16–17 h (B) before being immunostained for BTK (red) or p-BTK at DIV9. LPC treatment induced a demyelination (loss of GFP signal B, C) and a concomitant 8.5-fold increase in BTK signal (B, D). Similarly, p-BTK was also increased after LPC demyelination (E). (*p < 0.01; ***p < 0.0001). Scale bar = 100 μm.

Using the same method of quantification, we analyzed the calbindin expression (Fig. 3). Mean values of Calb⁺ area fraction (expressed in %) were obtained from images (n = 8) of cerebellum slices treated with BTKi (n = 4) vs control (n = 4) and report that the axon density is the same in BTKi treated conditions compared to control conditions (Fig. 3F). To assess the remyelination in cerebellar organotypic slices after BTKi treatment, images were acquired using an Olympus FV-1200 Upright Confocal Microscope. Z-series were performed at 0.3 μm increment, and green, red, and far-red fluorescence were acquired sequentially. Z-projections of images were carried out using Fiji software (NIH, Bethesda, Maryland). Images were taken from 5 slices per condition, 4–6 images per slice. At least a coverage of 60–250 internodes per slice, identified by PLP⁺ myelinated segments were counted (Fig. 3D), and the number of paranodes was determined by counting the number of Caspr⁺ structures for at least a coverage of 105–380 paranodes per slice (Fig. 3E). Results were expressed as mean±SEM of at least three independent experiments.

Inhibition of BTK favors remyelination. A–C: Cerebellar organotypic slices from P9 mouse, were exposed to LPC at DIV6 for 16–17 h. At DIV 9, i.e., peak of demyelination, myelinated internodes were no longer observed and only PLP + myelin debris were detected A). At the end of LPC-induced demyelination, cultures were treated with BTKi (BTKi-1) (1 μM) from DIV 9 before triple labeling with anti-PLP (myelinated internodes, green), anti-Caspr (paranodes, white) and anti-Calbindin (axons, red) antibodies. Small white arrows in control (Remyelinated-Ctrl B) and BTKi treated (Remyelinated-BTKi C) cultures point to some complete nodal structures with the Caspr⁺ paranode on each side of the Calbindin⁺ naked axon. D–F: Remyelination was evaluated by counting either PLP⁺ myelinated internodes (D) or Caspr⁺ paranodal structures (E) and axonal density as measured by the intensity of Calbindin labeling (F) (see M & M section for details). Scale bar = 10 μm