Figure 3. Molecular architecture and septate junctions in naïve and compressed nerves.

A1-3. Wild-type mice at 2-3-month-old age were perfused. Non-compressed sciatic nerves were dissected and teased into individual nerve fibers. Slides were stained with antibodies against MBP (blue) and Caspr (green). The former revealed internodal myelin (blue in A1). Caspr stained paranodes (arrows) that flanked the node of Ranvier. Caspr was also expressed at the Schmidt-Lanterman incisures (arrowhead in A2) and along the inner mesaxon as it spirals around the axon (arrow in A3). Voltage-gated potassium channels (Kv1.2; red in A3) were found in the juxtaparanodes (arrows in A3). Na_v were concentrated in the node of Ranvier (arrowhead in A3) and appeared as a narrow band. B-E was from non-compressed pmp22+/− nerves. Na_v, Kv1.2, Caspr and MBP were all localized in their proper regions. In addition, MAG was also correctly localized in the paranodes (arrows in D) and incisures (arrowhead in D) similar to what was observed in the wild-type myelinated nerve fibers. Tomacula were mainly found in the paranodal regions (B and E) and almost always extended beyond the paranodes and into juxtaparanodes and internodes (between arrows in B and E). F. EM was performed on the longitudinal section of a non-compressed pmp22+/− sciatic nerve. Normal paranodal septate junctions (arrowhead array) were observed in the region with tomacula. G. Compressed sciatic nerves from the second compression model (surgically exposed and clamped sciatic nerve) and non-compressed sciatic nerves (H) were sectioned into 10μm thickness and stained with antibodies against Caspr. Localization of Caspr in the paranodes (arrowheads in G and insets, and in supplementary Figure 1) was normal in compressed nerves. So was Kv1.2. However, axons revealed by YFP had smaller diameters and appeared stretched (G). Supporting this notion, we observed the typical undulating ‘wave’ appearance of axons in non-compressed nerve (asterisk array in H). However, these waves disappeared in the compressed nerve (see G), suggesting the compressed nerve was physically stretched during the compression. Notice that the intensity of axonal YFP was much weaker in the compressed nerves. This change has been very helpful for precisely defining the region of compression (please see supplementary Figure 1 for details). Within the compressed region, there were small islands of axons with strong intensity (arrows in 3G and supplementary Figure 1A) which likely were spared from compression forces. Insets: Caspr localization in paranodes was visualized under high power.