Acquired Neuropathies Figure 2. Ultrastructure of nodes of Ranvier in superficial peroneal nerves from 2 CIDP patients. A. A myelinated fiber (Patient 6) with a vacuolar inclusion (arrow) in the outer cytoplasm of the Schwann cell. B. A myelinated fiber (Patient 9) displaying numerous vacuoles in the cytoplasm of the Schwann cell (black arrows). C. Higher magnification showing a vacuole in the Schwann cell (arrow), and intact adherens junctions (white arrowhead) and septate-like paranodal axoglial junctions (black arrowhead) junctions. Note that paranodal loops are regularly spaced and tightly linked to the axolemma (black arrowhead). D. Another myelinated fiber from the same patient in which the Schwann cell cytoplasm contains large vacuoles with granular material (arrow), lamellar and multivesicular bodies (arrowheads). Scale bar: A, 0.6 mm; B, 3 mm; C, 300 nm; D, 1 mm. doi:10.1371/journal.pone.0014533.g002 In CIAP nodal regions, the abaxonal glial region and Schwann cell microvilli were normal (Figure S1). Some nodes were heminodes or were flanked by two Schwann cells with different degrees of myelination, some of which had very thin myelin (Fig. 3A–C) suggesting that they corresponded to myelination of regenerating fibers. The axoplasm was rich in organelles (Fig. 3C, arrows). In other fibers vacuoles of various sizes were frequently observed in the axoplasm (Fig. 3D–F). In the paranodal region vacuoles were found in the vicinity of the glial loops, often in regions with poorly defined axoglial junctions (Fig. 3F, arrow), whereas other regions displayed normal septate-like junctions (Fig. 3G arrow, Fig. S1C, arrowhead). The axolemma formed protrusions which contained cellular organelles, mitochondria, and vesicles, some of which possibly corresponded to glial invaginations (Fig. 3G, arrowhead). Thus, electron microscopy showed the existence of multiple abnormalities of nodal and paranodal regions in chronic acquired neuropathies. Alterations in paranodin and Nav immunoreactivities differ in CIDP and CIAP Since the nodal and paranodal regions were altered in CIDP and CIAP we examined the distribution of Nav that are located at nodes of Ranvier [22], and of paranodin, an axonal membrane glycoprotein highly enriched at paranodes [17] (Fig. 4). In the control nerve, Nav immunoreactivity was restricted to nodes of Ranvier (Fig. 4A, B, red) and flanked by paranodin immunore- activity, which had a thin extension along the internal mesaxon (Fig. 4A, B, green), as reported in rodents [17,23]. In CIDP, many fibers did not display the typical nodal/paranodal immunostaining described above since paranodin and Nav immunoreactivities had a punctuate appearance all along the axon (Fig. 4C, D), sometimes extending between two consecutive nodes (Fig. 4E). This diffuse distribution was apparent in samples from other patients (Fig. 5). In contrast, nodal regions appeared usually normal, some with particularly intense Nav immunofluorescence (Fig. 4C, D asterisk). In CIAP nerves, paranodin immunoreactivity was distributed irregularly at paranodes, with areas of weak or absent staining (Fig. 4F–I). Nav enrichment at nodes appeared normal, although a diffuse, internodal, punctiform Nav-like immunoreactivity of variable intensity was also detected in the 10 patients examined (Fig. 4G, H, arrowhead). Paranodes were sometimes interrupted (Fig. 4G, H, I, arrow) and heminodes (Fig. 4F, arrow) or asymmetrical nodes were observed, more frequently in CIAP than in CIDP (1861% of 230 nodes and 7.560.5% of 600 nodes, respectively). Other CIAP samples displayed a similar pattern of paranodin immunoreactivity, clearly different from that observed in CIDP (Fig. 5). KCNQ2 channels are altered in CIDP and CIAP We then examined the distribution of KCNQ2, a potassium channel subunit present in nodal regions [15]. As expected, KCNQ2 and Nav immunofluorescence were colocalized in nodal regions in control nerves (Fig. 6A). In the six CIDP nerves examined, the staining intensity of KCNQ2 was diminished or absent from nodes identified by Nav staining (Fig. 6B). In fibers displaying a diffuse punctuate distribution of Nav immunoreac- tivity, KCNQ2 immunoreactivity was also observed along the fibers (Fig. 6B). In eight CIAP nerves, KCNQ2 immunoreactivity colocalized with the nodal accumulations of Nav as in controls, but was also detected in internodal regions in some nerve fibers (Fig. 6C). Interestingly, wheras Nav and KCNQ2 immunoreac- tivities colocalized at nodes, it was not the case in the internodes. A mouse model of induced demyelination leads to alterations similar to those observed in CIDP In CIDP patients we observed an altered distribution of some neuronal proteins. Previous studies have shown that PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14533