Figure 2: In vivo imaging and correlated electron microscopy reveal myelinosome formation in acute neuroinflammatory lesions.

(a) In vivo image of an acute EAE lesion in a Thy1-YFP16/Biozzi ABH mouse revealing swollen axons and disrupted myelin sheaths in areas of cell infiltration (axons, white; nuclei, cyan; myelin, magenta throughout the figure). (b) In vivo time-lapse showing the formation of a myelin out-folding (‘myelinosome’) along an EAE axon. (c) Confocal image of a myelinosome stained for myelin (MBP) in a Thy1-YFP16/Biozzi ABH mouse perfused at 2 days after EAE onset. The graph shows the density of myelinosomes per mm axon at different time-points of EAE (healthy control, n=3 mice, 254 axons segments with 6.27 mm of total axon length were evaluated; Onset+2, n=6 mice, 449 axon segments with 15.8 mm of total axon length were evaluated; Onset of Progression+2, OnP+2, n=3 mice, 238 axon segments with 9.1 mm of total axon length were evaluated; Onset of progression+20, OnP+20, n=5 mice, 480 axon segments with 16.6 mm of total axon length were evaluated). (d) ‘Near infrared branding’ marks (dark lines) allow correlated ultrastructural reconstructions of myelinosomes (the boxed example is reconstructed in e and magnified in inset). (e) Three-dimensional rendering of the myelinosome shown in d. (f) Single ultrathin section through the centre of the myelinosome, showing the detachment of myelin (pseudocoloured magenta) from the axon, and inclusion of a myelin sphere inside (inset). (g–i) Electron micrographs from high-pressure frozen spinal EAE tissue illustrating the presence of myelinosomes that encompass all myelin leaflets (g), that show splitting of the myelin sheaths (h) and that contain internal myelin structures (i). (a) Scale bar, 50 μm; (b,c) Scale bar, 5 μm; (d) Scale bar, 20 μm (overview) and 10 μm (inset); (e) Scale bar, 1 μm (scale cube); (f (inset)) Scale bar, 200 nm; (g,h) Scale bar, 2 μm; (i) Scale bar, 1 μm.