Expression of 5-HT3 receptors and TTX resistant sodium channels (Na(V)1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders

Nikolaos Christidis, Isabell Kang, Brian E Cairns, Ujendra Kumar, Xudong Dong, Annika Rosén, Sigvard Kopp, Malin Ernberg, Nikolaos Christidis, Isabell Kang, Brian E Cairns, Ujendra Kumar, Xudong Dong, Annika Rosén, Sigvard Kopp, Malin Ernberg

Abstract

Background: Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls.

Methods: Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels.

Results: A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly higher in patients (42.6%) compared to healthy controls (12.0%) (P < .001).

Conclusions: This study showed that the 5-HT3A-receptor is highly expressed in human masseter and tibialis muscles and that there are more nerve fibers that express 5-HT3A-receptors in the masseter of women with myofascial TMD compared to healthy women. These findings indicate that 5-HT3-receptors might be up-regulated in myofascial TMD and could serve as potential biomarkers of chronic muscle pain.

Figures

Figure 1
Figure 1
Example of high power (40 x) photomicrographs for a masseter section from one female patient with myofascial TMD and a healthy age- and sex-matched pain-free volunteer. The fibers shown are positively labelled for PGP 9.5 (A), 5-HT3A(B), and NaV1.8 (C). (D) is the composite image. Calibration bar: 100 μm.
Figure 2
Figure 2
A low power (10 x) (in A) and a high power (40 x) (in B) image of the same section of a masseter muscle (microbiopsy from a pain-free volunteer). A. The arrows indicate individual muscle fascicles composed of myocytes separated by connective tissue. B. A higher power (40 x, rounded square in A) image of PGP 9.5 labelling with examples of fibers associated with myocytes (arrows) and connective tissue (double arrows). The insets show examples of a PGP 9.5 positive fiber associated with a myocyte (i) and with connective tissue (ii). Calibration bar A: 100 μm, B: 25 μm.
Figure 3
Figure 3
A comparison showing the different mean frequencies for antibody labelling on PGP 9.5 immunoreactive fibers (5-HT3A; both 5-HT3A and NaV1.8) in the masseter and tibialis tissues. There were no significant differences in the frequency of fibers that expressed 5-HT3A or both 5-HT3A and NaV1.8, when the two muscles were compared (P > .05, Students t-test). Each bar represents the average from all the sections obtained from each muscle type. The error bars depict standard error of the mean.

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