TRPM8 Axonal expression is decreased in painful human teeth with irreversible pulpitis and cold hyperalgesia

Abstract

Pulpitis pain might be triggered by a cold stimulus, yet the cellular mechanisms responsible for this phenomenon are largely unknown. One possible mechanism involves the direct activation of cold-responsive thermoreceptors. The purpose of this study was to evaluate the possible role of the TRPM8 thermoreceptor in cold-mediated noxious pulpal pain mechanisms by comparing expression patterns in pulpal nerves from healthy control molars to cold-sensitive painful molars with irreversible pulpitis. Samples were identically processed with the indirect immunofluorescence method, and images were obtained with confocal microscopy. The immunofluorescence intensity and area occupied by TRPM8 within N52/PGP9.5-identified nerve fibers were quantified. Results showed that relative to normal samples, TRPM8 nerve area expression was significantly less in the cold-sensitive painful samples (34.9% vs 8%, P <0.03), but with no significant difference in immunofluorescence intensity between the 2 groups. These results suggest that TRPM8 is most likely not involved in cold-mediated noxious pulpal pain mechanisms.

Figure 1

Confocal micrographs of combined TRPM8 (red), N52 (green) and PGP9.5 (blue) stainings (left side; A and C) and the corresponding TRPM8 image alone (right side; B and D) in the coronal pulp of a normal sample (A, B) and a painful sample (C, D). (A, B) Normal sample; TRPM8 expression is seen within a subset of larger and medium-sized N52/PGP9.5 identified nerve fibers (arrowheads), while lacking in the smaller axons, such as those that are seen in the odontoblastic layer (O; arrows). (C, D) Painful sample; TRPM8 (red) expression is generally lacking in the N52/PGP9.5 identified nerve fibers (arrows) located adjacent to inflammatory cells associated with a carious lesion, including the nerve fibers that traverse the odontoblastic layer (O). All scale bars = 100 μms.

Figure 2

Confocal micrographs of combined TRPM8 (red), N52 (green) and PGP9.5 (blue) stainings (left side; A and C) and the corresponding TRPM8 image alone (right side; B and D) in axon bundles located in the upper radicular pulp of a normal sample (A, B) and a painful sample (C, D). (A, B) Normal sample; TRPM8 (red) expression is seen both on the surface and within the axoplasm of the larger N52/PGP9.5 identified nerve fibers (large arrows), while some of the smaller fibers express TRPM8 (small arrows) and others do not (arrowhead). The TRPM8 expression on the surface of the larger fibers that extends beyond the N52/PGP9.5 staining is seen as a “halo” and most likely represents the staining of myelin. (C, D) Painful sample; TRPM8 (red) is weakly expressed in some of the larger fibers (arrows), while the smaller fibers generally lack expression (arrowhead). All scale bars = 20 μms.

Figure 3

Image analysis results obtained for each sample (N = normal and P = painful). (A) The percent of N52/PGP9.5 identified nerve fiber area occupied by TRPM8 within each sample. (B) The average immunofluorescence intensity of TRPM8 staining within each sample as based on 12 bit images with a 0–4095 range. Error bars represent standard error of the means.