Study Design and Ethics Statement
This study was designed as a randomized, sham-controlled, and double-blinded trial with repeated measures within subject design at the Medical University of Graz, Austria. After receiving approval from the ethics committee (EK 30-270 ex 17/18), healthy male and female subjects aged 20–60 willing to participate were included. Exclusion criteria were defined as: known allergies or hypersensitivity to capsaicin, history of skin diseases, neurological diseases, diabetes mellitus, pregnancy, and consummation of analgetic or psychotropic drugs taken within 1 month before study participation.
Those eligible were informed about the study details by the principal investigator and, after giving written consent, were included in the study. During the first visit, demographic information, including Fitzpatrick classification of skin color, was obtained from all patients .
A-Priori Sample-Size Calculation
No previously published data were available as a source for an a-priori sample-size calculation. Thus, we powered the study to meet the following parameters: the significance level of 0.05 and beta was 0.2. In a two-sided paired t test, a sample of ten participants is suitable for detecting an effect size of ≥ 1.
The trial took place in a quiet room with an average temperature of ~ 24 °C over the whole duration of the capsaicin application and irradiation procedure. Probands were situated in the room for at least 30 min to allow acclimatization.
Capsaicin Pain Model
We used a human pain model capable of inducing the central and peripheral mechanisms. Capsaicin application rapidly produces local neurogenic inflammation (characterized by edema and erythema) when locally administered to human skin by stimulating the TRPV1 receptors on dermal sensory nerve endings [5, 16,17,18,19,20]. In this pain model, heat hyperalgesia (i.e., the increased sensitivity to heat stimulation at the site of application of capsaicin) has been found to be due to peripheral sensitization, whereas mechanical hyperalgesia (i.e., increased sensitivity to mechanical stimulation beyond the area of capsaicin) is due to central sensitization .
Before the capsaicin application, skin temperature was measured using an infrared thermometer. In the case of skin temperature below 30 °C, the area was covered by a blanket until the required skin temperature was reached. A 30 mm × 30 mm cutaneous capsaicin patch 8% (Qutenza, Grünenthal, Aachen, Germany) with 640 mg of capsaicin per cm2 (corresponding to 5.76 mg of capsaicin in total) was applied in the volar side in the middle of both distal forearms of the participants. The patches were loosely fixed with bandages. After 60 min, the patches were removed, and the exposed skin areas were treated with the appropriate cleansing gel (supplied with Qutenza and intended for removal) according to the manufacturer’s recommendations.
Randomization and Blinding
During the capsaicin application, patients were randomized to determine which arm was treated with LLLT. The other arm received the same treatment with an unactivated device and served as sham-control. Randomization was carried out by a coin toss (left or right) by a nurse, who had no further involvement in the study except for the irradiation procedure. The observer was not present during the randomization and intervention. The nurse was instructed not to share any information about the allocation of the side of irradiation with the volunteers and observers.
The volunteers were blinded with opaque goggles. In addition, they wore earplugs to prevent them from hearing the ventilator noise of the LLLT system.
Immediately after removing the capsaicin patch, the two forearms were positioned in the same way on a base divided by an opaque screen to avoid scattered radiation (Fig. 2). The nurse, who was also responsible for the randomization, positioned two identical LLLT devices (Repuls7, Repuls Lichtmedizintechnik GmbH, Vienna, Austria) simultaneously with a 7 cm-distance ring in direct contact with the skin. According to the randomization procedure, one LLLT device remained turned off. The other forearm was irradiated with pulsating and cold red light of 640 nm. The intensity was 175 mWcm−2, which corresponds to a power density of 4100 mW. The pulse frequency was set to 2.5 Hz. A duration of 12 min was chosen based on the manufacture’s recommendations.
Measurement was performed directly after intervention. Pain intensity was quantified using a numeric rating scale (NRS 0–10) immediately after intervention on the irradiated and non-treated forearms. Assessment of the sensory thresholds was based on the specifications of the German Research Association for Neuropathic Pain [22, 23].
HPT was assessed on both the LLLT irradiated forearm and non-treated forearm (= control). It was evaluated using a computer-controlled Peltier element, the TSA-II Quantitative NeuroSensory Analyzer (Medoc Ltd., Ramat Yishai, Israel), with a 30 mm × 30 mm thermode. As heat hyperalgesia is only present in the area of primary hyperalgesia , the probe of the TSA was exactly positioned to match the marked edges of the capsaicin patch. The baseline temperature of the probe was 32 °C. The ramp for the temperature increase of the probe was set to 1 °C/s. The volunteers were instructed to press a button as soon as they felt a sensation that was not only warm but also accompanied by a burning, stinging, drilling, or dragging feeling.
MPT was assessed on both sides 1 cm proximal to the marked area using pinpricks with a calibrated force of 8, 16, 32, 64, 128, 256, and 512 mN and a flat contact area 0.2 mm in diameter (MRC Systems GmbH, Heidelberg, Germany). The pinpricks were applied with a stepwise which increased force. The volunteers were instructed to indicate the force where they first perceived a sharp sensation. They were then asked about any side effects at the end of the irradiation and the end of the measurement.
The data were analyzed for normality by the Shapiro–Wilk test. Accordingly, a comparison of the sides was calculated using a paired t test or Wilcoxon signed-rank test, as appropriate. The significance level α was set to 0.05. To quantify the magnitude of the effect size, Cohen’s d for repeated measurement was calculated with their corresponding 95% confidence interval (95% CI). The effect sizes were summarized in three categories: small (0.2 < d < 0.5), medium (0.5 < d < 0.8), and large (d > 0.8) .
The force measurements of the mechanical pain sensitivity were transformed into decadic logarithms. Before the logarithmic transformation, a constant of 0.1 was added to all zero and non-zero raw data in order to avoid a loss of zero values . The data were summarized as mean and standard deviations.
All analyses were conducted with NCSS [NCSS 12 Statistical Software (2018); NCSS, LLC. Kaysville, UT, USA]. Data analyses were performed by an independent researcher blinded to the group assignment.
This manuscript was prepared according to the Consort guidelines. All procedures were in accordance with the Helsinki Declaration.