Twelve participants aged between 18 and 80 years were recruited (50% women, mean age = 31 ± 15; Table 1). The exclusion criteria were history of chronic pain or/and neurological or psychiatric diseases, peripheral nervous system injury or neuropathy, diseases causing potential neural damage (e.g., diabetes, diseases of the immune system, oncological diseases), previous clinical history of cervical surgery, injuries or surgery affecting the upper limb, altered sensitivity in the tested regions, frequent headaches, and/or orofacial pain.

Each subject was invited to participate in a single testing session that lasted approximately 1 h. Sensory testing was conducted in a quiet indoor laboratory at ambient temperature (22°C–26°C). The subjects sat in a comfortable chair and were familiarized with the experimental procedure. Participants were prohibited from undertaking vigorous physical activities or drinking caffeinated beverages at least 24 h before the experiment.

The session started with baseline testing with stimuli (TS, see below) applied at the C5, C6, and C7 dermatomes at the dominant side, and at the contralateral C6 dermatome at 5 min intervals for 15 min (see Figure 1 and TS section below). The cutaneous electrical HFS was then applied to the C6 dermatome at the dominant side during 60 s (see HFS section below), and the subject was asked to rate the electrical pain intensity during the electrical stimulus. After HFS, TS were applied to the C5, C6, and C7 dermatomes at the dominant side, and at the contralateral C6 dermatome to detect homotopic and heterotopic effects at 5 min intervals for 45 min. Thus, the study compared post HFS effects on sensory and pain intensity with the 15 min baseline recordings made from the same participant. Subjects were blinded to the study hypothesis.

The presence of neuropathic characteristics related to pain and induced by HFS was assessed with the seven items of the DN4 questionnaire: burning, painful cold, electric shocks, tingling, pins and needles, numbness, and itching (29). The participants rated the intensity of every neuropathic sensation using the NRS (0: absence; 10: maximum intensity) before and up to 45 min after the HFS. Thus, these assessments were performed at 5, 10, and 15 min before the HFS, and 5, 10, 15, 20, 25, 30, 35, 40, and 45 min after the HFS.

The Patient Health Questionnaire-9 (PHQ-9) and the 7-item generalized anxiety disorder (GAD) scale (GAD-7) were self-report instruments used to assess depression and anxiety, respectively, at baseline.

The PHQ-9 is a self-report screening test that includes nine items related to diagnostic criteria for major depression. The presence of depressed mood, anhedonia, sleep problems, feelings of tiredness, changes in appetite or weight, feelings of guilt or worthlessness, difficulty concentrating, feelings of sluggishness or worry, and suicidal ideation over the previous 2 weeks period were assessed with this questionnaire. Items are scored on a four-point Likert scale from 0 (never) to 3 (most days). Thus, total severity scores range from 0 to 27 points indicating moderate (10–14 points), moderately severe (15–19 points) and severe (20–27 points) levels of depressive symptoms (30, 31).

The GAD-7 includes 7 items related to anxiety symptoms rated on a 4-point response scale ranging from 0 (“never”) to 3 (“nearly every day”). The total severity score ranges from 0 to 21 points indicating minimum (0–4), mild (5–9), moderate (10–14) and severe (15–21) anxiety (32, 33).

Health-related quality of life was assessed with the paper-based five-level version of EuroQol-5D (EQ-5D-5L), which assesses independent daily activities, mobility, self-care, perceived pain, and depression/anxiety impact domains. All responses, ranging from 1 (absence of problems) to 3 (severe problems), were converted into a single index number, which corresponds to the health state according to standardized values ranging from 0 (health state equivalent to death) to 1 (optimal health) (34, 35). Additionally, this questionnaire also includes a visual analogue scale, where general health during the previous 24 h is rated on a scale from 0 (worse imaginable health) to 100 (best imaginable health) (35). This assessment was performed at baseline.

Data analysis was conducted using Sigma Plot version 11.0 (Systat Software, Inc., United States) and SPSS version 22.0 (IBM Corp., Armonk, New York, United States). Results are expressed as mean, standard deviation (SD), and standard error (SE). In general, the Shapiro–Wilk test revealed that quantitative data, except for TS sensitivity, followed a normal distribution (p > 0.05). Measures of TS sensitivity were transformed into percentages; the scores of 1 to 3 were transformed to 33%, 66% and 100%, respectively. The repeated measures analysis of variance (RM-ANOVA) test followed by post-hoc pairwise tests using the Holm-Sidak correction was performed in order to investigate change in TS sensation, pain intensity following HFS, or pain intensity during HFS (trend of pain across time). General differences for TS sensations or pain intensity, and neuropathic sensations before and after HFS were calculated with the paired student’s t-test. The interaction between dermatomal test site and time after HFS on TS sensation or pain intensity was analyzed with the two-way ANOVA. Additionally, the possible influence of baseline demographic or clinical characteristics on HFS-induced changes in sensation or pain intensity were analyzed with the χ² tests of independence, unpaired student t-test, and one-way ANOVA followed by post-hoc pairwise tests to control for multiple comparisons. Finally, the Pearson correlation (r) test was used to evaluate associations between variables. The statistical analysis was conducted at a 95% confidence level with a p-value <0.05 which was considered statistically significant.

Twelve healthy subjects (50% women, mean age = 31 ± 15 years) were recruited. Clinical and demographic data are presented in Table 1. No significant differences in baseline characteristics were identified for age or sex, except for body mass index (BMI; p = 0.034): overweight individuals (BMI: 25.0–29.9) revealed higher PHQ-9 scores compared to normal weight (BMI 18.5–24.9) (7 ± 3.5 VS 3.86 ± 1.2).

No visible skin injuries occurred following the electrical HFS, although skin blushing under the stimulated area was observed. Figure 2A shows pain ratings perceived during the HFS. The 100 Hz conditioning stimulation induced an increasing pain intensity during the HFS (temporal summation), i.e., when compared to the initial pain intensity rated with HFS value, a significant increase in pain intensity was perceived from 20 s up to 60 s.

Figure 2A shows the mean (and SE) perceived pain intensity elicited during the HFS at ipsilateral C6 dermatome (dominant side; see Figure 1A). A significant increase in pain intensity was observed from 20 to 60 s (Figure 2A). The pain intensity perceived during the electrical HFS was significantly and positively associated with age (r = 0.716; p = 0.009): older individuals reported higher pain intensity during the electrical stimulus (Figure 2B). Individuals older than 40 years old perceived higher pain intensity during HFS compared to those subjects younger than 30 years old (8 ± 1 vs. 4.5 ± 1.9, p = 0.02). Additionally, a significant negative association was observed between pain intensity during HFS and perceived quality of life (r = −0.633; p = 0.027): subjects who reported poor quality of life perceived higher pain intensity with HFS (Figure 2C). No other significant differences in age range were found (p > 0.05) and no significant differences in BMI were revealed (p > 0.05).

Single electrical test applied stimuli at the ipsilateral C6 and contralateral C6 dermatomal test sites before and after electrical HFS revealed significant depotentiation of pain intensity (Figure 2D). A main effect of time was revealed: a significant decrease in perceived pain intensity was identified for the ipsilateral C6 dermatomes at 45 min and contralateral C6 test site at 45 min after HFS (p < 0.01). No significant potentiation was observed at 20 min after HFS for the ipsilateral test site. The percentage change in ipsilateral C6 pain intensity at 45 min after HFS is shown in Figure 2E.

Table 2 shows changes in electrical pain sensitivity reported by each participant at 5–15, 20–30 and 35–45 min after HFS: 75% of healthy individuals showed depotentiation between 35–45 min after HFS; however, 50% of participants demonstrated potentiation at 20–30 min interval after HFS.

During the 45 min after HFS, a significant increase in the number of neuropathic sensations were reported (0.6 ± 0.7 vs. 1.0 ± 0.8; p = 0.023), and especially when measured between 15 and 45 min after HFS (0.6 ± 0.7 vs. 1.0 ± 0.9; p = 0.018). The greatest change in frequency of neuropathic sensations was associated with tingling sensation: after HFS, the presence of tingling sensation tripled when compared in the pre-HFS interval (Figure 2F). No other significant changes were found.

Figure 3 reveals changes in mechanical sensitivity after HFS at the ipsilateral C6 dermatome test site (dominant side). No change in sensitivity was identified with brush (Figure 3A). In contrast, a significant increase in mechanical pain sensitivity (von Frey filament) at ipsilateral C6 dermatome (Figure 3B) was identified from 5 and up to 45 min after HFS. No other significant changes or significant differences among sites were found.

Figures 3, 4 reveal changes in thermal sensitivity after HFS at ipsilateral C6 and C7 dermatomes, and contralateral C6 dermatome. For cold sensitivity (Figure 3C), a significant main effect of time was revealed: a significant increase in cold sensitivity was found at the ipsilateral C6 dermatomes from 10 up to 40 min after HFS (Figure 3D). In addition, an increase in contralateral cold sensitivity at the C6 dermatome at 25 min after HFS was also identified. Additionally, some individuals also presented low pain intensity (NRS: 1–3) related to cold hypersensitivity after HFS. No other significant changes were found in the ipsilateral C5 or C7 dermatomes.

Significant sex differences were found in cold sensitivity induced after HFS at C6 dermatome (dominant side), particularly between 15 and 45 min (p = 0.02): women showed higher cold sensitivity than men (3.0 ± 0.0 vs. 2.5 ± 0.8). Additionally, significant sex differences were found in mechanical pain sensitivity (pinprick) between 5 and 45 min after HFS at the C6 dermatome (dominant side, p = 0.045), particularly between 15–45 min (p = 0.036): women showed higher mechanical pain sensitivity than men (0.2 ± 0.04 vs. 0.0 ± 0.0). No other significant sex differences were found and no significant associations with levels of anxiety or depression were revealed.

Electrical HFS increased the perception of neuropathic sensations, especially tingling as the most common neuropathic descriptor. Previously found hot and burning sensations were reported as the most common neuropathic descriptors (23), which were related to perceived pain intensity at the HFS site. These sensations have been attributed to the activation of Aδ-fibers and C-fibers (23).

This study also showed a significant increase in mechanical pain sensitivity at the HFS-conditioned site supporting similar findings observed around the HFS-conditioned site in previous studies (2, 3, 8), possibly mediated by Aδ fibers (3, 10, 13) and primary nociceptive C-fibers (3, 4, 12, 23, 36). Furthermore, long-lasting mechanical hypersensitivity to pinprick stimuli for more than 45 min has been reported previously (2), with an increase in pinprick-evoked brain potentials (37). Although the present findings should be considered with caution due to the small sample size, the results of the current study support the involvement of LTP in the mechanical sensitization at the conditioned site following electrical HFS.

After HFS, significant sex differences in mechanical and cold pain sensitivity were also revealed for the first time: women showed higher mechanical and cold pain sensitivity than in men. A previous study found no significant sex differences on HFS-induced pain sensitivity (13), although women showed a greater increase in mechanical pain sensitivity after electrical stimulation (13) and in pain sensitivity in general (38). Although the present findings need to be considered with caution due to the small sample size and further studies are needed to understand possible mechanisms underlying sex differences, pain perception during HFS could help to identify risk factors which underlie the development of chronic pain (39). This includes the possible association of pain perception during HFS with age and health-related quality of life.

The current findings revealed a significant increase in cold sensitivity in both the ipsilateral and contralateral C6 dermatomes after HFS, unlike previous studies that reported no significant changes (14). The significant increase in cold sensitivity at the conditioned site could indicate a possible role for Aδ-fibers in cold sensitivity and hyperalgesia (40, 41) and thermal hyperalgesia at the stimulated area is a typical feature of primary hyperalgesia associated with this sensitization (4, 42). The observation of cold sensitivity in the contralateral dermatome could be explained that lumbar Lamina I neurons also receive contralateral afferent information (43), which may be disinhibited following periods of intense pain stimulation such as that applied with the electrical HFS stimulus (6, 7). This mechanism would lead to contralateral sensory information to projection neurons contributing to thermal hypersensitivity and mirror-image pain. Indeed, observations of cold hyperalgesia in participants with unilateral carpal tunnel syndrome have been observed (44) and quantitative sensory testing has revealed evidence for contralateral changes in sensitivity with different pain pathologies (6, 7). Similarly, HFS could also trigger LTP of unconditioned C-fiber synapses through glial cell processes (16). After HFS, no significant change in heat sensitivity was revealed supporting previous studies (2, 4, 14, 45). Nevertheless, the present findings should be considered with caution due to the small sample size.

The current study also found a significant depotentiation in electrical pain sensitivity at the conditioned and contralateral sites, supporting a concurrent reduction of the perceived pain intensity after HFS at the conditioned site and several remote test sites (5, 9–11), including the contralateral test area (5, 9, 10). Initial studies of HFS on electrical test stimuli revealed a long-term potentiation of pain intensity elicited by single electrical stimuli at the HFS site (1–3, 8, 13).

HFS could activate hypoesthesia induced by continuous electrical stimulation at C-fiber strength (4, 46). HFS could also reverse LTP with the induction of long-term depression (LTD) or LTP-induced depotentiation, depending on neuronal properties such as the membrane potential of spinal dorsal horn neurons (9), the type of cell activated demonstrated as LTP in spinothalamic neurons and LTD at GABAergic neurons (2, 47), and finally the implication of glia (48). Although the present findings should be considered with caution due to the small sample size, the recent observations of inhibition of electrical pain intensity at several test sites after HFS suggest that careful selection of test stimulus modality is critical to understanding processes of LTP and LTP-induced depotentiation in pain modulation.

The small participant sample size requires some caution to be made in the interpretation and generalization of results from the present study. Nevertheless, sample size was based on sample sizes used in previous studies (3, 14), which even included smaller sample sizes (3). Furthermore, to our knowledge, this is the first study that has identified long-lasting changes in cold sensitivity after HFS at the conditioned site and contralateral test site. Further study of cold sensitization following HFS using quantitative sensory testing protocols may provide an important insight into the development of persistent pain mechanisms. In addition, the association of pain intensity experienced during HFS with age and quality of life, and the sex effect of induced pain sensitivity after HFS warrants further investigation into the influence of these demographic and clinical characteristics on LTP and pain sensitization in humans.