Sodium channel Nav1.6 accumulates at the site of infraorbital nerve injury

Abstract

Background: Sodium channel (NaCh) expressions change following nerve and inflammatory lesions and this change may contribute to the activation of pain pathways. In a previous study we found a dramatic increase in the size and density of NaCh accumulations, and a remodeling of NaChs at intact and altered myelinated sites at a location just proximal to a combined partial axotomy and chromic suture lesion of the rat infraorbital nerve (ION) with the use of an antibody that identifies all NaCh isoforms. Here we evaluate the contribution of the major nodal NaCh isoform, Nav1.6, to this remodeling of NaChs following the same lesion. Sections of the ION from normal and ION lesioned subjects were double-stained with antibodies against Nav1.6 and caspr (contactin-associated protein; a paranodal protein to identify nodes of Ranvier) and then z-series of optically sectioned images were captured with a confocal microscope. ImageJ (NIH) software was used to quantify the average size and density of Nav1.6 accumulations, while additional single fiber analyses measured the axial length of the nodal gap, and the immunofluorescence intensity of Nav1.6 in nodes and of caspr in the paranodal region.

Results: The findings showed a significant increase in the average size and density of Nav1.6 accumulations in lesioned IONs when compared to normal IONs. The results of the single fiber analyses in caspr-identified typical nodes showed an increased axial length of the nodal gap, an increased immunofluorescence intensity of nodal Nav1.6 and a decreased immunofluorescence intensity of paranodal caspr in lesioned IONs when compared to normal IONs. In the lesioned IONs, Nav1.6 accumulations were also seen in association with altered caspr-relationships, such as heminodes.

Conclusion: The results of the present study identify Nav1.6 as one isoform involved in the augmentation and remodeling of NaChs at nodal sites following a combined partial axotomy and chromic suture ION lesion. The augmentation of Nav1.6 may result from an alteration in axon-Schwann cell signaling mechanisms as suggested by changes in caspr expression. The changes identified in this study suggest that the participation of Nav1.6 should be considered when examining changes in the excitability of myelinated axons in neuropathic pain models.

Figure 1

Na_v1.6 and caspr staining. A and B. Confocal micrographs from average-intensity z-projections (45 slices in A; 47 slices in B) demonstrate Na_v1.6 (red) and caspr (green) staining relationships seen within the normal (A) and lesioned (B) ION. A. The normal ION shows bright Na_v1.6 staining (arrowhead) at nodes of Ranvier that are identified as the gap seen between the paranodal staining of caspr. The caspr staining is intense within the paranodal region and prominent Na_v1.6 staining is generally restricted to the narrow area at the node. B. The lesioned ION, at a location just proximal to the chromic suture, shows a change in the Na_v1.6 and caspr staining relationships that were seen in A. These changes include a loss in the overall organization and the immunofluorescence intensity of caspr staining within the paranodal region (arrowheads) and an increased size and intensity of Na_v1.6 within accumulations (large arrows). Alterations in the normal Na_v1.6 and caspr staining relationships include Na_v1.6 accumulations that show an association with caspr on one side (heminodes; small arrow). Both images were collected with the same laser settings. Scale bars = 10 μm.

Figure 2

Na_v1.6 and caspr staining. A and B. Confocal migrographs of Na_v1.6 (red) and caspr (green) staining relationships seen within individual fibers in the normal (A) and lesioned (B) ION. A. Individual fibers within the normal ION show bright Na_v1.6 staining (arrowhead) at nodes of Ranvier that are identified as the gap seen between the dense paranodal staining of caspr (arrow). B. Individual fibers within the lesioned ION show alterations in the Na_v1.6 and caspr staining relationships that include "heminodes" (small arrowheads) and "split nodes" that vary in the distance seen between the Na_v1.6 accumulations (small and large arrows). Some large Na_v1.6 accumulations are flanked on both sides by reduced caspr staining (large arrowheads), while other nodes appear more normal (asterick). Scale bars = 5 μm.

Figure 3

Results of quantitative analyses performed in normal (N) and lesioned (L) infraorbital nerves. A and B. The density (A) and average pixel size (B) of Na_v1.6 accumulations (with a size of ≥ 15 pixels) was significantly greater in lesioned IONs than seen in normal IONs. C. The average immunofluorescence intensity (0–65,535 range) of pixels with Na_v1.6 staining located in caspr-identified typical nodes was significantly greater in lesioned IONs when compared to normal IONs. D. The average axial length (in pixels) of the nodal gap within caspr-identified typical nodal accumulations was significantly greater in lesioned IONs when compared to normal IONs. E. The average immunofluorescence intensity (0–65,535 range) of pixels with caspr staining located within the paranodal regions of caspr-identified typical nodes was significantly greater in normal IONs when compared to lesioned IONs.