Figure 1.
Transverse semi-thin sections of the musculo-cutaneous nerve of a control and patients with chronic acquired neuropathies.
A. Normal density of myelinated fibers in a nerve without lesion. B. In a CIDP nerve (Patient 12), several hypomyelinated fibers (arrowheads) and an onion bulb formation (arrow) are visualized. C. In CIAP nerve (Patient 13), loss in myelinated fibers and numerous regenerating clusters (arrowheads) are present. Scale bar 30 µm.
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 µm; B, 3 µm; C, 300 nm; D, 1 µm.
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 µm, B and E: 20 nm, D, E: 10 µm, C, F, and G: 300 nm.
Figure 4.
Alterations of nodal and paranodal proteins in chronic acquired neuropathies.
Immunolocalization of paranodin (Pnd, green), voltage-gated Na+ channels (Nav, red) in longitudinal sections of superficial peroneal nerves (A–I). In blue is shown nuclear staining with topro-8 (E, I) and neurofilaments (Nf, F). B, D, and H are higher magnifications of A, C, and G, respectively. A, B. In a control nerve paranodin and Nav are correctly localized in the paranodal (arrows) and nodal (asterisks) regions, respectively. C, D. In CIDP (Patient 1), paranodin immunoreactivity extends to the internodal regions (double arrows). Nav immunoreactivity remains concentrated in some fibers (asterisk), while it has a diffuse, punctuate pattern along other fibers (arrow). E. Another CIDP case (Patient 6) with paranodin immunostaining (double arrows) extending in the internode. Nodal regions are indicated (asterisks). F. In CIAP (Patient 14), in some fibers the paranodal staining appears irregular (arrow). Neurofilaments are in blue. G, H. In CIAP patient 20, paranodin immunoreactivity is irregular in some nerve fibers with an asymmetrical localization (arrow), whereas the nodal expression of Nav appears normal (asterisk). A diffuse Nav-like immunoreactivity can be detected along some axons (arrowhead). I. In another CIAP Patient 13, an interrupted paranodal region (arrow) is visible at high magnification, followed by the normal paranodin staining along the mesaxon. Scale bar: A, C, E, F, G, 40 µm; B, D, H, 20 µm; I, 1.7 µm.
Figure 5.
Comparison of paranodin immunoreactivity in several patients with CIDP and CIAP.
Longitudinal sections from superficial peroneal nerves were immunostained with antibodies for paranodin in four patients with CIDP (A) and four patients with CIAP (B). The patient numbers are indicated on the figure. In CIDP samples paranodin immunoreactivity was visible along the internode in most fibers (A, arrows). In contrast in CIAP paranodin was restricted to the nodes (B, arrows), which appeared often asymmetrical or irregular. Scale bar 20 µm.
Figure 6.
KCNQ2 immunoreactivity in a control nerve and in patients with CIDP and CIAP.
Immunolocalization of KCQN2 (green) and voltage-gated Na+ channels (Nav, red) in longitudinal sections of superficial peroneal nerves. A. KCNQ2 and Nav immunoreactivities colocalize at the nodal region in the control nerve (arrowheads) and are not detected in the internodes. B. In a CIDP nerve (Patient 5), nodal KCNQ2 immunoreactivity is low (asterisk) or absent (arrowhead) and only a few nodes are labeled. A weak immunoreactivity is observed for the two ion channels in some internodes. C. In a CIAP sample (Patient 18), KCNQ2 and Nav colocalization is observed at nodes (arrowheads) but some immunoreactivity is also detected along the fibers. Scale bar: 20 µm.
Figure 7.
Distribution and levels of paranodin are increased in sciatic nerves in a mouse model of demyelination induced by conditional Krox 20 inactivation.
Sciatic nerve sections were prepared 28 d after a 5-day treatment with vehicle (A) or tamoxifen (B) in Krox20lacZ/flox, R26-CreERT mice and immunolabeled for paranodin (Pnd, green) and Nav (red). A. In vehicle-injected animals, paranodin immunoreactivity is concentrated at paranodes (arrow) on both sides of the nodal Nav accumulations (asterisk). B. In tamoxifen-injected animals, paranodin immunoreactivity extends outside of the paranodal regions. In a fiber in which the position of the node is indicated by an asterisk, paranodin immunoreactivity is visualized all along the internodal region (arrowheads). In A and B two magnifications are shown. Scale bar: 45 µm for the upper panels and 20 µm for the lower panels. C. Western blot analysis of sciatic nerves extracts using an anti-paranodin antibody. Adult brain was analyzed for comparison. Alpha-tubulin was used for normalization of the quantity of protein. D. Quantification of immunoblot data obtained from sciatic nerves of 3 different mice (1 nerve per mouse) and in which paranodin immunoreactivity is normalized to alpha-tubulin. Statistical analysis with Student t-test.
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 µm.