Acquired Neuropathies Figure 8. Distribution of voltage-gated Na+ channels (Nav) and KCNQ2 is altered in sciatic nerves of a mouse model of demyelination induced by conditional Krox 20 inactivation. Sciatic nerve sections were prepared 28 d after vehicle (A, C) or tamoxifen (B, D) injections in Krox20lacZ/flox, R26-CreERT mice. A. B. Immunolabeled for Nav (green) and phosphoneurofilament (Nf, antibody SMI31, red). In vehicle- injected animals, Nav expression is restricted to nodes (asterisks) (A). In tamoxifen-injected animals, Nav immunostaining is visible in nodal regions (asterisk) and, in some fibers, all along the axon (arrow) with a punctiform pattern (B). C, D. Longitudinal sciatic nerve sections were also doubly stained with antibodies for KCNQ2 (green) and Nav (red). In control vehicle-injected animals (C), KCNQ2 and Nav immunoreactivities colocalized at nodes of Ranvier (arrowhead). D. In tamoxifen-injected animals, KCNQ2 immunoreactivity is low and only a few nodes are co-stained (arrowhead) while in others KCNQ2 immunostaining is weak or absent (asterisks). Scale bar: 20 mm. doi:10.1371/journal.pone.0014533.g008 In CIDP, by contrast, paranodin immunofluorescence was increased and much more diffuse than in normal nerves. These modifications are likely to result directly from the Schwann cells alterations revealed by electron microscopy, since the correct localization of paranodin to the paranodal regions depends on myelinating glial cells [25,26]. In central myelinated axons from patients with multiple sclerosis, demyelination results in a diffuse expression of paranodin [35] and its levels appear downregulated [36]. Delocalization of paranodin was also observed in Charcot- Marie-Tooth skin biopsies [21] and in dysmyelinating mutant mice [20,25]. However, in contrast to these various conditions in which paranodin levels appeared normal or decreased, we found an increased paranodin immunoreactivity in CIDP, in addition to its delocalization. Nav expression appeared also increased in CIDP nerves and diffusely distributed in internodal regions of some axons. This is likely to result from the loss of axo-glial contact in demyelinated axons [37]. To further investigate the alterations of nodal and paranodal proteins induced by acquired demyelination, we studied mice in which Schwann cells dedifferentiation can be induced in adult animals [27]. These mice provide a unique model in which the genetically targeted and pharmacologically induced disappearance of a single transcription factor results in the disappearance of compact myelin. In contrast with toxic chemicals used for inducing demyelination there is neither Schwann cell death nor lesions of the axons. This model allows a specific investigation of the consequences of Schwann cells dedifferentiation on axonal proteins. As in CIDP, we observed an increased and diffuse paranodin immunolabeling with a diffusion of Nav immunoreac- tivity in some fibers. Increased levels of paranodin protein were demonstrated by immunoblotting, in contrast to what has been observed in dysmyelinating mutants [20,25]. It is not known whether this corresponds to an increased production and/or a diminished destruction of the protein. Interestingly, we observed an increased density of ankyrin G clusters, presumably corre- sponding to nodes of Ranvier. This observation is likely to be related to the proliferation of Schwann cells reported in mice with tamoxifen-induced Krox20 deletion [27]. This would result in shorter internodes with an increased density of nodes or protonodes. In fact some nodes of Ranvier visualized by ankyrin G or Nav immunoreactivity appeared well preserved, in agreement with their slower alterations as compared to paranodes following dysmyelination [20,25,26]. In contrast, other nodes displayed a decreased KCNQ2 and Nav labeling, which may correspond to newly formed nodes attesting to the ongoing remyelination. Since paranodin immunofluorescence is a robust marker of paranodes, differently altered in CIDP and CIAP, we tested whether it could be used as a help to the sometimes difficult diagnosis of these neuropathies. Using very simple criteria based on the intensity and distribution of paranodin immunoreactivity, we found that unaware observers were able to classify correctly PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e14533