Nav1.7 expression is increased in painful human dental pulp

Songjiang Luo, Griffin M Perry, S Rock Levinson, Michael A Henry, Songjiang Luo, Griffin M Perry, S Rock Levinson, Michael A Henry

Abstract

Background: Animal studies and a few human studies have shown a change in sodium channel (NaCh) expression after inflammatory lesions, and this change is implicated in the generation of pain states. We are using the extracted human tooth as a model system to study peripheral pain mechanisms and here examine the expression of the Nav1.7 NaCh isoform in normal and painful samples. Pulpal sections were labeled with antibodies against: 1) Nav1.7, N52 and PGP9.5, and 2) Nav1.7, caspr (a paranodal protein used to identify nodes of Ranvier), and myelin basic protein (MBP), and a z-series of optically-sectioned images were obtained with the confocal microscope. Nav1.7-immunofluorescence was quantified in N52/PGP9.5-identified nerve fibers with NIH ImageJ software, while Nav1.7 expression in myelinated fibers at caspr-identified nodal sites was evaluated and further characterized as either typical or atypical as based on caspr-relationships.

Results: Results show a significant increase in nerve area with Nav1.7 expression within coronal and radicular fiber bundles and increased expression at typical and atypical caspr-identified nodal sites in painful samples. Painful samples also showed an augmentation of Nav1.7 within localized areas that lacked MBP, including those associated with atypical caspr-identified sites, thus identifying NaCh remodeling within demyelinating axons as the basis for a possible pulpal pain mechanism.

Conclusion: This study identifies the increased axonal expression and augmentation of Nav1.7 at intact and remodeling/demyelinating nodes within the painful human dental pulp where these changes may contribute to constant, increased evoked and spontaneous pain responses that characterize the pain associated with toothache.

Figures

Figure 1
Figure 1
Nav1.7 expression in pulp horn. A-F. Confocal micrographs of single optical images showing Nav1.7 (red) expression within N52 (green) and PGP9.5 (blue)-identified nerve fibers within the pulp horn of one normal (A, B) and two painful (C-F) samples. A and B. Nav1.7 (arrow) is expressed in many nerve fibers in the normal pulp horn (A) that stain with N52 and PGP9.5 as seen in the combined image in B. C and D. The pulp horn from a painful sample also shows Nav1.7 expression (arrow) in most nerve fibers that stain with N52 and PGP9.5 as seen in the combined image in D. E and F. The pulp horn of one painful sample contains no Nav1.7 staining (E) within fibers that appear fragmented as visualized with N52 staining (F; arrows). Scale bars = 50 μm.
Figure 2
Figure 2
Nav1.7 expression in axon bundles. A-D. Confocal micrographs of single optical images showing Nav1.7 (red) expression within N52 (green) and PGP9.5 (blue)-identified nerve fibers within axon bundles in normal (A, B) and painful (C, D) samples. A and B. Nav1.7 (red; arrows) is expressed in nerve fibers that appear intact as visualized with PGP9.5 and especially N52 (green) staining within an axon bundle located in a normal sample (A), while a different normal sample shows some fibers that appear fragmented as seen with N52 staining (B; arrow). C and D. The expression of Nav1.7 within a painful sample (C) appears prominent within intact nerve fibers (arrows), but less in fibers that appear fragmented (arrowheads) as visualized with N52 staining. A different painful sample contains many fibers with prominent Nav1.7 staining, including some that show minimal staining with N52 and PGP9.5 (D; arrow). Scale bars = 20 μm in A-C and 100 μm in D.
Figure 3
Figure 3
Results of quantitative analyses of Nav1.7 expression within nerve fibers in normal and painful samples. A-F. The nerve area and pixel intensity of Nav1.7 expression within N52/PGP9.5-identified nerve fibers was quantified within different regions in normal and painful samples. A and B. The nerve area (A) and pixel intensity (B) of Nav1.7 expression was not significantly different within the pulp horns of normal and painful samples. C-F. The nerve area of Nav1.7 expression was significantly greater within coronal (C) and upper radicular (E) axon bundles in painful samples, but with no difference between normal and painful samples in pixel intensity within these same regions (D, F). ** = p < 0.01, *** = p < 0.001.
Figure 4
Figure 4
Nav1.7 expression at caspr-identified nodal sites. A-D. Confocal micrographs of maximum-intensity z-projections (five z-sections with spacing increments of one μm) showing the expression of non-thresholded (A, B) and thresholded (C, D) Nav1.7 (red) immunofluorescence at caspr (green)-identified sites within axon bundles in normal (A, C) and painful (B, D) samples. Typical nodal forms (arrows) predominate in the normal sample (A, C) and one of these shows Nav1.7 expression located within the nodal gap (arrowhead). The painful sample (B, D) shows atypical nodal forms that include both heminodes (black arrows) and a split node (arrowhead). Many of these atypical nodal forms and some of the typical nodal forms (white arrows) are associated with Nav1.7. E and F. The expression of Nav1.7 at typical and atypical caspr-identified nodal sites was determined within axon bundles located in coronal and radicular regions in normal and painful samples. Results show a significant increase in the percent of both typical and atypical nodes that were associated with Nav1.7 within the coronal (E) and radicular (F) axon bundles in painful samples. G and H. All caspr-identified nodal sites were evaluated within axon bundles located in coronal and radicular regions in normal and painful samples and were classified as either typical or atypical. Results show a significant increase in the percent of atypical nodal sites within the coronal (G) and radicular (H) axon bundles in painful samples. Scale bars = 20 μm; * = p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Nav1.7 expression at caspr-identified nodal sites as related to state of myelination. A-C. Confocal micrographs of collapsed z-projection images (five z-sections with spacing increments of one μm) showing Nav1.7 (red), caspr (green) and myelin basic protein (MBP; blue) staining relationships in one normal (A) and two painful (B and C) samples. A and B. The normal sample (A) shows the expression of MBP and caspr associated with some myelinated fibers (arrows), while this expression is absent in other smaller fibers that most likely lack myelin (arrowheads). The expression of Nav1.7 is prominent within the fibers that lack myelin. In contrast, the painful sample seen in (B) contains many fibers with prominent Nav1.7 expression at caspr-associated nodal sites (white arrows), but that show either alterations (arrowheads) or a lack of MBP staining altogether (black arrows). C. Combined and single channel images of Nav1.7, caspr, and MBP staining relationships seen in a painful sample showing the increased expression of Nav1.7 within an axon segment that is flanked on both sides with caspr and that lacks MBP (arrows), whereas Nav1.7 expression is less in the areas of this same axon where MBP expression is present (arrowheads). Scale bars = 20 μm.

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