Local gene expression changes after UV-irradiation of human skin

Abstract

UV-irradiation is a well-known translational pain model inducing local inflammation and primary hyperalgesia. The mediators and receptor proteins specifically contributing to mechanical or heat hyperalgesia are still unclear. Therefore, we irradiated buttock skin of humans (n = 16) with 5-fold MED of UV-C and assessed the time course of hyperalgesia and axon reflex erythema. In parallel, we took skin biopsies at 3, 6 and 24 h after UVC irradiation and assessed gene expression levels (RT-PCR ) of neurotrophins (e.g. NGF, BDNF, GDNF), ion channels (e.g. NaV1.7, TRPV1), inflammatory mediators (e.g. CCL-2, CCL-3) and enzymes (e.g. PGES, COX2). Hyperalgesia to mechanical impact (12 m/s) and heat (48 °C) stimuli was significant at 6 h (p<0.05 and p<0.01) and 24 h (p<0.005 and p<0.01) after irradiation. Axon reflex erythema upon mechanical and thermal stimuli was significantly increased 3 h after irradiation and particularly strong at 6 h. A significant modulation of 9 genes was found post UV-C irradiation, including NGF (3, 6, 24 h), TrkA (6, 24 h), artemin, bradykinin-1 receptor, COX-2, CCL-2 and CCL-3 (3 and 6 h each). A significant down-regulation was observed for TRPV1 and iNOS (6, 24 h). Individual one-to-one correlation analysis of hyperalgesia and gene expression revealed that changes of Nav1.7 (SCN9A) mRNA levels at 6 and 24 h correlated to the intensity of mechanical hyperalgesia recorded at 24 h post UV-irradiation (Pearson r: 0.57, p<0.04 and r: 0.82, p<0.001). Expression of COX-2 and mPGES at 6 h correlated to the intensity of heat-induced erythema 24 h post UV (r: 0.57, p<0.05 for COX-2 and r: 0.83, p<0.001 for PGES). The individual correlation analyses of functional readouts (erythema and pain response) with local expression changes provided evidence for a potential role of Nav1.7 in mechanical hyperalgesia.

Figure 1. Pain ratings (VAS, 0–100) upon (A) mechanical and (B) heat stimuli.

Mechanical impact stimuli were delivered at 12 m/s and continuous heat applied for 5 sec at 48°C. Responses recorded at 3, 6 and 24 h post irradiation are depicted in open columns from untreated skin and those recorded from UV-C treated skin (5-fold MED) in black columns. Asterisks indicate significant differences between control and UV-C skin (p<0.01, Fishers LSD test).

Figure 2. Time course of skin erythema development (cm²) upon (A) mechanical and (B) heat stimuli.

Responses to mechanical impact stimuli (12 m/s) and continuous heat (5 sec at 48 C) were recorded from untreated control (open circles) and UV-C treated skin (5-fold MED, solid squares) at 3 h (left), 6 h (middle) and 24 h (right panel) post irradiation. The hatch indicates significantly increased blood flow 24 h after UV-C irradiation (p<0.0001, Fishers LSD test), filled triangles indicate the time of stimulation (m: mechanical, h: heat), and asterisks indicate significant differences between control and UV-C skin (p<0.0001, Fishers LSD test). Note that heat tests were performed post mechanical stimulation that had caused slightly increased baseline perfusion.

Figure 3. Group analysis of mRNA expression increase (RT-PCR, n = 14).

Changes upon UV-C irradiation are depicted as fold increase above baseline (dashed horizontal line represents no change) assessed at 3, 6 and 24 h post irradiation. Error bars indicate the 95% confidence interval.

Figure 4. Correlation analysis of mRNA expression (n = 14) and 24 h hypersensitivity upon (A) heat and (B) mechanical impact.

Individual one-to-one correlation analyses of functional hypersensitivity 24 h post UV-C irradiation and mRNA expression changes at 6 h (left panel) and 24 h (right panel). All values were normalized to control skin. (A): Correlation of relative increase of cyclooxygenase 2 (COX-2, upper panels) and prostaglandin synthase (mPGES, middle panels) expression with relative increase of heat-evoked erythema (48°C, 5 sec) recorded at 24 h after irradiation. (B): Correlation of NaV1.7 (gene SCN9A) expression changes at 6 h (left) and 24 h (right) after UV-C irradiation with relative increase of mechanical impact pain (12 m/s) recorded 24 h post irradiation. Significant correlations (p<0.05, Bonferroni corrected) are marked by asterisks.