Quantitative Sensory Testing Of Periauricular Skin In Healthy Adults
Study participants Twenty healthy young adults participated in the study. According to the gender, they were divided into two groups: 10 women (mean age ± SD: 23.2 ± 0.8 years; range: 22–25 years) and 10 men (mean age ± SD: 23.1 ± 1.1 years; range: 21–25 years). All the participants were healthy without orofacial pain complaints or symptoms of pain in head, face, and neck regions. Exclusion criteria were: history of trauma in the orofacial area that interfered with normal somatosensory function, any acute or chronic orofacial diseases (for example, burning mouth syndrome, trigeminal neuralgia, chronic headache, systemic musculoskeletal pain disorders such as fibromyalgia, or symptoms of rheumatoid arthritis, etc.), use of medication such as muscle relaxants, anticonvulsants, antidepressants, or anxiolytics within the last month, and severe systematic diseases or mental disorders. The study was conducted in accordance with the guidelines set forth in the Declaration of Helsinki II. Declaration and informed consent were obtained from all participants prior to participation. The study was approved by the Nanjing Medical University Research Ethics Committee with NO: PJ 2018-040-001.
Experimental protocol The experiment was performed in a quiet room with the temperature controlled between 21–25 degree Celsius. At the beginning of the experiment, all participants were informed about the purpose, content and method of this experiment, and confirmed that they fully understood. All tests were performed by the same tester using the same instrument and the tests were repeated by the same tester after one week.
The following QST parameters were tested by a method of limits: cold detection threshold (CDT), warm detection threshold (WDT), cold pain threshold (CPT), heat pain threshold (HPT), mechanical detection threshold (MDT), mechanical pain threshold (MPT), pressure pain threshold (PPT) and two-point discrimination (2PD). All parameters were measured at four sites innervated by the great auricular nerve (GAN): bilateral subauricular sites (LA, RA), bilateral postauricular sites (LB, RB) and one site innervated by the radial nerve: the dorsum of the left hand (control) (Fig. 1)17. In addition, a standardized palpometer (PalpeterR Sunstar Suisse) with a 0.5 kg force was used to apply pressure stimulation at 9 bilateral points at each of the subauricular and postauricular sites. Figure 1 Test sites for QST: bilateral subauricular sites (A), bilateral postauricular sites (B), the surface of left hand (C). Thermal detection and thermal pain thresholds Thermal quantitative sensory tests were performed with the use of a computerized thermal stimulator (MEDOC TSA-2001 apparatus, Medoc Ltd, Ramat-Yishai, Israel)18,19. The contact area of the thermode was 30 × 30 mm. CDT, WDT were measured first, followed by CPT, HPT. The temperature of the thermode started at a baseline of 32 °C and cooled down or heated up at a rate of 1 °C/s to the lower limit of 0 °C or upper limit of 55 °C. Participants were instructed to press a button on the computer mouse as soon as they perceived the thermal sensation of cold, warm, cold pain, or heat pain. Then, the procedure ended, and the temperature returned to baseline. The mean thresholds of three consecutive measurements were calculated.
The verbal instructions given to the participants were in accordance with the QST guidelines as specified by the German Neuropathic Pain Network (DFNS)5. Mechanical detection and mechanical pain thresholds MDT was measured with the use of standardized Semmes–Weinstein monofilaments with 20 different diameters (North Coast Medical, Canada). The number of each filament (1.65–6.65) corresponds to a logarithmic function of the equivalent forces of 0.008–300 g. To detect the MPT, weighted pinprick stimuli delivered with a custom-made set of seven pinprick stimulators (Aalborg University, Denmark) were used. Each stimulator had a flat contact surface of 0.2 mm that exerted forces of 8–512 mN20. MDT and MPT were determined by the method of limits, and defined as the geometric mean of 3 series of descending and ascending stimulus intensities5. Pressure pain threshold A handheld pressure algometer (Algometer, MEDOC, Israel) with a probe diameter of 1.0 cm was used to test the pain sensitivity to stimuli applied to test sites by a qualified dentist who was specifically trained according to the DFNS examination protocol5. The algometer was applied vertically to the test sites and the applied pressure was linearly increased according to the computer prompt (30 kPa/sec). The participant was instructed to press a hold switch connected to the computer as soon as the sensation of non-painful pressure changed to a sensation of pain. The PPT value was then determined from the display. Three trials were made in random order at each test sites. There was of 1 min interval between each measurement to minimize sensitization and/or habituation to the stimulus. The mean threshold of three measurements was calculated. Two-point discrimination (2PD) A vernier caliper was used to vertically contact the two tips with a distance of 15 mm to the test site, the participant was asked to judge if the stimulus was perceived as one or two points. If two points was reported, the distance between the tips was reduced by 1 mm each time until the participant reported only one point, then the threshold (distance between tips) was recorded. Three threshold measurements were made and the mean was used for further statistical analysis. Mechanical sensitivity mapping A quantitative mechanical palpometer (PalpeterR, Sunstar Suisse SA company was used for standardized palpation in the bilateral subauricular and postauricular sites. Each of the subauricular and postauricular sites was divided into 3 × 3 grids. The palpometer with 0.5 kg force was applied to the 9 grids in randomized order13. All test points were stimulated for approximately 2 seconds during each measurement. After each measurement, there was a 10-second interval for the participant to rate the perceived intensity of the stimulus on a 0-50-100 numerical rating scale (NRS), in which 0 means no sensation, 50 means just barely painful, 100 means most pain imaginable21. In one session, both sides of the subauricular and postauricular sites were tested three times and the average NRS score of the three stimuli was calculated for each measurement point. In order to make sure that the same test points were measured, a standard template was fabricated to identify the test sites prior to the test, consisting of 2 squares (3*3 cm) (A、B). Each of the 2 squares was divided into 9 squares, the side of which was 10 mm (Fig. 2a). The center of the square A is on the line between the otobasion inferius (Obi) and the gonion (Go), and the Obi is at the midpoint of the superior edge of the square A (Fig. 2b). The 18 points were marked on the skin of subauricular and postauricular sites with a marker pen in accordance with the standard template (Fig. 2c). Figure 2 Test points for mechanical sensitivity mapping. (a) A standard template was fabricated to identify the test sites prior to the test, consisting of 2 squares (3*3 cm) (A,B). Each of the 2 squares was divided into 9 squares, the side of which is 10 mm. (b) Each of the subauricular and postauricular sites was divided into 3 × 3 grids. The center of the square A is on the line between the otobasion inferius (Obs) and the gonion (Go) and the Obi is at the midpoint of the superior edge of the square A. (c) The 18 points were marked on the skin of subauricular and postauricular sites with a marker pen in accordance with the standard template. Statistical analysis All data were tested for normality distribution before further statistical analysis and log converted if not normally distributed. Intra-class correlation coefficients (ICC) and a 95% confidence interval (CI) were presented to analyze the consistency of the thresholds and NRS values over days. The value of ICC is between 0 and 1. An ICC less than 0.4 represents a poor agreement, an ICC of 0.4–0.59 is regarded fair agreement,an ICC of 0.6–0.75 is considered good agreement, and an ICC more than 0.75 indicates an excellent agreement. Besides, the standard error of measurement (SEM) and its 95% confidence interval, the smallest real difference (SRD) were calculated (standard error of measurement = intra-individual standard deviation * √(1-ICC), SRD = 1.96* standard error of measurement *√2). The standard error of measurement indicates the expected error between two measurements conducted under the same circumstances in the same subject over a defined period of time. The lower the standard error of measurement, the better the test-retest-reliability23. One-way analysis of variation (ANOVA) was used to test for differences in the thresholds and NRS scores over days with a significance level of 5%. Two-way ANOVA was used to test differences in the thresholds with the following factors: gender (2 levels),
test sites (4 levels). Three-way ANOVA was used to test differences in NRS scores with the following factors: gender (2 levels), test sites (4 levels) and test points (9 levels). Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study.
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