It was estimated that a group size of 12 healthy men and 12 healthy women would permit the identification of a difference of 30% (± 25%) in nerve fiber expression of NR2B receptors, with a power of 0.80, and alpha set at 0.05. Moreover, it has been previously determined that significant sex-related differences in experimental pain can be identified with groups of 12 or more are investigated 25,26. However, fifteen healthy female participants and fifteen age-matched healthy male participants (mean±SD age: 30±12 years) were recruited by ads posted on the internet-page “www.forsoegsperson.dk” as well as at Aarhus University, Denmark. None of the participants had taken anti-inflammatory or analgesic medication within 24 hours of the procedure. Pregnancy or facial pain, palpatory tenderness, neurological disorder, inflammatory diseases, fibromyalgia, whiplash-associated disorders, and neuropathic disorders, were considered as exclusion criteria. To ensure that the results are not affected by the influence of muscle tenderness on palpation, participants were screened for TMD and orofacial pain complaints using diagnostic criteria for TMD (DC/TMD) 1. Participants received informed consent before inclusion. The ethical committee Aarhus approved the experiment (Midtjylland, approval No. 1-10-72-199-15), which followed the guidelines of the Helsinki declaration.
The study consisted of 3 sessions at days 0, 7, and 10 and was based on a published technique for injections with glutamate and NGF 27. On day 0, 1 M of glutamate (0.2 mL sterile solution; Skanderborg Apotek, Aarhus, Denmark) was injected on the experimental side (left side). On day 7, 0.4 mL NGF (25 μg/mL sterile solution; Skanderborg Apotek, Aarhus, Denmark) was injected on the same side. A one week wash-out period was used to avoid any possible effect of an interaction of NGF and glutamate on the muscle afferent fibers. Microbiopsies were obtained from the masseter on the control side on day 0 and on the experimental (injection) side on day 10. Pain intensity at rest was recorded directly after the injections and during the following 5 min. Mechanical sensitivity, such as pressure pain threshold (PPT), temporal summation, as well as chewing pain and fatigue (chewing gum for 1 min) were recorded respectively from both sides in each session, as well as before (as a baseline) and 5 min after the injections.
Assessments of experimental pain induced characteristics
Pain intensity after glutamate or NGF injection was recorded during 5 min on an electronic visual analog score (eVAS) ranging from 0 to 10. The lower endpoint of the eVAS was marked with “no pain” while the upper endpoint was marked “worst pain imaginable”. The highest eVAS score (peak pain) was calculated from the recorded eVAS data and used for further analysis.
Pressure pain threshold
PPT (KPa), the first perceived painful amount of pressure, was recorded from two sites over each masseter muscle, the injection point and a point 1 cm superior to the injection point using an electronic algometer (Somedic Sales AB, Hörby, Sweden)28 with a 1 cm2 rubber covered tip that was placed perpendicular to the skin-surface overlaying the muscle. The increase rate in pressure was 30 kPa/s. The mean of three recordings that were made over each site was used for analyses.
Temporal summation and chewing test
Temporal summation was assessed with 1.0 kg Palpeter (Sunstar Suisse SA, Etoy, Switzerland)29, by repeated applications of mechanical pressure for 1 second with a 2-second interval for ten times 30. During temporal summation and chewing test 30, participants were asked to rate their pain level on a 0 - 10 numerical rating scale (NRS) and their fatigue level on Borg’s ratings of perceived exertion (RPE) scale (6 - 20) 31.
A technique developed by Christidis and co-workers in 2014, to obtain enough amount of muscle tissue from an equivalent region within the muscle overall participants 32 was used when taking microbiopsies. First, topical anesthesia (EMLA Patch®, 25mg lidocaine and 25 mg prilocaine, AstraZeneca, Södertälje, Sweden) was applied for half an hour, over the skin surface covering to the most prominent part of the masseter muscle during contraction. Then a co-axial needle within a guiding instrument (Bard®TruGuide™; BARD Norden, Helsingborg, Sweden) was inserted with an angulation of 45 degrees, 1 cm below the zygomatic arch, along the near long axis of the muscles until the fascia was penetrated to a marked depth of 10 mm. After that, the needle was removed, while the instrument remained in place. Finally, a biopsy instrument (Monopty®Bard®) with a penetration depth of 11 mm and a diameter of 18G, was inserted through the guiding instrument to collect the masseter muscle biopsy 33.
Immunohistochemistry and picture analysis
A blinded analysis was performed by a researcher who did not collect or code the microbiopsies. After obtaining samples, the microbiopsies were fixed over-night at 4°C, with 4% paraformaldehyde, rinsed in phosphate-buffered saline (PBS), dehydrated, then frozen in a -80 freezer. On the day of staining, sections with a thickness of 10 μm were incubated in normal donkey serum (ABCAM Inc, Cambridge, England, ab7475) for 1 hour, and then for 24 hours with primary antibodies against the specific axonal markers: (A) 1:250; anti-PGP 9.5 antibody, ABCAM Inc, Cambridge, England; ab72911; (B) 1:200; anti-NMDAR2B antibody, ABCAM Inc, Cambridge, England; ab65783; (C) 1:1000; anti-SP antibody, ABCAM Inc, Cambridge, England; ab10353; and (D) NGF 1:20; Human beta-NGF Affinity Purified Polyclonal Ab, R&D Systems Inc, 614 McKineley PL NE Minneapolis, AF-256-NA). Sections were rinsed with PBS and incubated with fluorescent secondary antibodies: (1) Alexa 488 donkey-anti-mouse, 1:700 for PGP 9.5; (2) Alexa Fluor 546 donkey anti-rabit, 1:700 for NR2B; (3) Alexa Fluor 633 donkey anti-goat, 1:700 for NGF, ThermoFisher, Burlington, ON, Canada; and (4) Alexa Fluor405 donkey anti-guinea pig, 1:700 for SP, Sigma-Aldrich, MO, USA). Removal of the primary antibodies was used as an approach to test antibody specificity. To analyze the sections as well as to capture images, a Leica TCS SPE Confocal Microscope (Leica microsystems, Wetzlar, Germany) was used.
The image processing and analysis program ImageJ (Image Processing and Analysis in Java; National Institutes of Health, USA) was used to detect and count PGP 9.5 positive nerve fibers, to calculate their area and to detect fibers (PGP 9.5) colocalization with different markers (SP, NR2B and NGF) 33. PGP 9.5 positive nerve fibers were either associated with myocytes (semi-round or tubular, well-defined cells with multiple nuclei at the periphery) or found within connective tissue (irregular tissue containing dense or loose fibers surrounding the myocytes) 34 (Fig. 1). A slide containing biopsies not used in immunohistochemistry was stained before analysis with Hematoxylin (HTX) to distinguish myocytes from connective tissue. Nerve fibers were considered positive if the PGP 9.5 fluorescent signals exceeded the mean background of the picture + 2 standard deviation (SD) and had a minimum length and width of 4 μm 12. If signals were separated by 5 µm or less, however, sharing the same path and tissue were considered expressed from the same fiber. The density was calculated by normalizing the PGP 9.5 positive counts to the area of the tissue present in the images (density= number of positive fibers in a tissue divided by the total area in square millimeters of the same tissue on an image and averaged over the number of images for each participant). The expression frequency was calculated using the following formula: number of PGP 9.5 positive fibers that were colocalized with other markers divided by the total number of PGP 9.5 positive fibers in the image and averaged over the number of images for the participant. More than half of all SP expressing neurons are C-fibers and most of these fibers are nociceptors 35. In the present study, fibers expressing SP were considered as putative nociceptors. The immunohistochemical analysis is presented in detail in a previously published study 33.
SigmaPlot for Windows version 14.0 software (Systat Software Inc., San Jose, CA, USA) was used for data analysis.
The difference in the mean peak pain intensity between glutamate and NGF was analyzed with paired t-tests. The sex differences in the peak pain after each injection were analyzed with t-tests. Normalized PPT (Post-injection PPT data divided by the baseline data, multiply by 100) was analyzed with a two-way repeated-measures (2-way RM) analysis of variance (ANOVA) with factors time (Day 0, 7 and 10) and sex. The 2-way RM ANOVA was followed by post hoc comparisons with the use of the Bonferroni test. For the other parameter (temporal summation, chewing pain, and fatigue), data were not normally distributed (Shapiro-Wilk), so RM ANOVA on Ranks, factor: time (Day 0, 7 and 10), with Holm-Sidak method as a post-hoc test was used, while the Mann-Whitney U-test was used to test sex differences.
In a previous study 33, it was shown that there was a significant difference in the expression of putative afferent sensory fibers between biopsies that contained mostly myocytes as compared to biopsies that contained mostly connective tissue. Hence, only participants whose biopsies containing the same tissue (myocytes or connective tissue) on both days were included in the analysis. The actual number of participants with tissues, regardless of its coexistence on both days, is also presented in Table 1. Pooling data from connective tissue and myocytes were intentionally avoided to prevent the risk of having an effect of one tissue on the other. For example, women on day 0 had more connective tissue than myocytes; hence pooling the data will bias the results to whatever is the fibers expression within the connective tissue on that day.
To detect significant differences and interaction between factors (day and sex) in the density and expression frequency of nerve fibers, a parametric 2-way RM ANOVA test was used and followed by post hoc comparisons with the use of the Bonferroni test. Due to the lack of samples containing myocytes in women on day 0, sex differences for myocytes are not reported.
Descriptive data are presented as mean ± standard deviation (SD) or median (IQR) depending on the distribution. The Spearman test was used to examine the correlation between the changes in parameters after glutamate injection and the expression of NR2B by nociceptive fibers, and also to test correlations between the change in parameters and the change in nerve fibers expression frequency after NGF injection. For all tests, the level of significance was set to P<0.05.