Figure 4

Demyelinated cerebellar white matter in taiep rats has second harmonic generating cells and a higher fluorescence intensity from neurofilaments. (A–F) Representative SHG micrographs from the cerebellum of WT (A–C) and mutant (D–F) rats. All animals tested gave identical results. The images are maximum projections from 5 planes separated by 7 μm. (G–L) Confocal images of stained neurofilaments from the cerebellum of WT (G–I) and mutant rats (J–L). (B, E, H and K) are zoomed regions in the molecular layer and (C, F, I, and L), in the cerebellar white matter, where the difference in signal distribution and intensity is better appreciated. In (F), arrow, cell bodies; star, intense rounded structure and dotted arrow, fiber-like structure. The calibration bar in panel (D), with brighter colors corresponding to the highest signal intensities, is also valid for (A, C and F) while the calibration bar in (E) is also valid for (B). The direction of laser polarization for SHG microscopy coincides with the scale bar orientation. (M) Schematic of cerebellar folia. (N, O) Representative line graphs of the intensity levels detected along the lines shown in panels A and D for SHG images, G and J for NF fluorescence, respectively. In SHG images, the WM of taiep rats shows the highest gray level values of the three layers. The mutant rat also displays higher WM NF fluorescence than the fluorescence from granular and molecular layers (n = 3 TUBB4A rats and 3 WT). (P) Gray levels histograms obtained from second harmonic signal of the cerebellar layers (2 WT and 3 taiep rats). (Q) NF fluorescence intensity in the WM of mutant rats was higher than the fluorescence from control rats, no significant difference was found in molecular and granular layers between WT and taiep (n = 3 taiep and 3 WT rats, *p < 0.05, two-tailed Mann–Whitney test). The average power used for SHG imaging was 13 mW.