Figure 3. Ultrastructure of nodes of Ranvier in superficial peroneal nerves from 2 CIAP patients. A. Low magnification of a fiber (Patient 14) with a short internodal (IN) distance between two consecutive heminodal regions (arrowheads), possibly corresponding to a regenerating fiber. B. Higher magnification of the left heminode in A showing the thinly myelinated segment (arrowhead). C. Higher magnification of B. Note the presence of numerous organelles in the axoplasm (arrows). D. Low magnification of a longitudinal ultrathin section of a node of Ranvier (NR) region (Patient 20). E. Higher magnification of D showing axoplasmic vacuoles (arrows). F. Higher magnification of E showing paranodal junctions (arrowhead) and a vacuolar space close to the axolemma (arrow). G. Another detail of E (bottom right) showing paranodal loops with axoglial junctions (arrow) and axoplasmic protrusions with organelles (arrowhead). Scale bar: A and D: 2.5 mm, B and E: 20 nm, D, E: 10 mm, C, F, and G: 300 nm. doi:10.1371/journal.pone.0014533.g003 dysmyelination alters axonal domains organization, with the disappearance of paranodal accumulation of proteins [24,25,26]. In these studies no overall increase in paranodin immunoreactivity was described, in contrast to what was observed in patients with acquired CIDP. In order to better understand how the disappearance of differentiated Schwann cells alters axonal proteins in peripheral nerves, we used a mouse model in which demyelination is induced in the adult by a mechanism which targets specifically the Schwann cells [27]. This model is based on the tamoxifen-induced deletion of the Krox20 gene, which codes for a transcription factor required for the maintenance of the expression of Schwann cell-specific genes. Induced demyelination in adult mice leads to the presence of macrophages degrading Schwann cells, onion bulbs, and abnormally thin myelin sheaths [27], which are reminiscent of alterations found in patients with CIDP. We examined paranodin and ankyrin G expression a month after tamoxifen treatment (Fig. 7). In vehicle-treated mice, paranodin and ankyrin G immunostaining were restricted to the paranodal and nodal regions, respectively (Fig. 7A). Paranodin Acquired Neuropathies immunofluorescence ended rather abruptly at the borders of the paranodal regions (Fig. 7A, arrow), with the exception of the labeling along the mesaxon. In tamoxifen-treated mice the limits of paranodin distribution were less regular and immunostaining extended into the internodal regions (Fig. 7B). Ankyrin G immunostaining was present in the nodal region but the density of these accumulations along the nerve fibers was higher in tamoxifen-treated (95 103610 103 nodes per mm3 mean 6 SEM, n = 3) than in vehicle-treated mice (38 10369 103 nodes per mm 3 , n = 3, t-test p,0.05). To determine whether the increased immunofluorescence corresponded to increased levels of proteins, we used immunoblotting (Fig. 7C). The levels of paranodin were increased in sciatic nerve extracts from tamoxifen-injected mice, as compared to mice only injected with vehicle (Fig. 7C, D), showing that the induced demyelination increased the expression of paranodin. The distribution of Nav sodium channels was examined in double labeling experiments using a polyclonal anti-pan Nav antibody and the mouse monoclonal antibody SMI 31, which reacts with a phosphorylated epitope in neurofilaments H and, to a lesser extent, M. In vehicle-injected mice Nav immunoreactivity was concentrat- ed in nodal regions identified as the narrowed region of the nerve fiber stained with SMI31 (Fig. 8A). In tamoxifen-treated mice Nav diffuse punctuate immunostaining extended all along some axons in which phosphoneurofilament labeling was very faint (Fig. 8B, arrow). We also compared the distribution of KCNQ2 and Nav in this mouse model. As expected, in vehicle-injected mouse KCNQ2 and Nav immunofluorescence were colocalized at nodes (Fig. 8C). In the tamoxifen-treated mice the staining intensity of KCNQ2 was diminished or absent in some nodes¸ while others were normal (Fig. 8D, arrowhead). A faint punctuate background immunoreac- tivity was also observed along fibers, which may indicate a diffuse distribution of KCNQ2. Paranodin immunofluorescence is a good marker for diagnosis of CIDP and CIAP Since paranodin immunostaining appeared to be consistently different between the two types of chronic neuropathies (see Figs. 4 and 5), we tested whether it could be used as a tool for their differential diagnosis. For this purpose we used the 17 samples in which the sections had not been already used for the above studies. Randomly mixed images from 9 CIDP and 8 CIAP nerve sections stained with paranodin antibodies were submitted to four observers unaware of the diagnosis, used to examine immunofluorescence images but untrained for this specific task. They were requested to utilize the following criteria: 1/ For CIDP: high global intensity of paranodin immunostaining, and its extension beyond the paranodes (mesaxon excluded); 2/ For CIAP: low global intensity and/or interruption and/or presence of hemi-nodes. Only longitudinally well-oriented fibers were analyzed leading to the exclusion of one case which was considered to be non-interpretable. The percentage of correct diagnosis using these two criteria for the 16 interpretable samples ranged from 63% to 82% (mean 6 SEM: 7064). The percent agreement between at least three independent observers was 70%. These results show that examination of paranodin immunoreactivity on a single section provides a good indication for the diagnosis of CIAP vs CIDP. Discussion The diagnosis of CIDP can be difficult and requires a combination of criteria, which includes in atypical cases histopath- ological features [5,8,10], in order to differentiate it from CIAP. In CIDP, demyelinating features include macrophage-mediated PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e14533