Electrically evoked neuropeptide release and neurogenic inflammation differ between rat and human skin

Abstract

Protein extravasation and vasodilatation can be induced by neuropeptides released from nociceptive afferents (neurogenic inflammation). We measured electrically evoked neuropeptide release and concomitant protein extravasation in human and rat skin using intradermal microdialysis. Plasmapheresis capillaries were inserted intradermally at a length of 1.5 cm in the volar forearm of human subjects or abdominal skin of rats. Capillaries were perfused with Ringer solution at a flow rate of 2.5 or 1.6 microl min(-1). After a baseline period of 60 min capillaries were stimulated electrically (1 Hz, 80 mA, 0.5 ms or 4 Hz, 30 mA, 0.5 ms) for 30 min using a surface electrode directly above the capillaries and a stainless-steel wire inserted in the capillaries. Total protein concentration was assessed photometrically and calcitonin gene-related peptide (CGRP) and substance P (SP) concentrations were measured by enzyme-linked immunosorbent assay (ELISA). In rat skin, electrical stimulation increased CGRP and total protein concentration in the dialysate. SP measurements showed a larger variance but only for the 1 Hz stimulation was the increased release significant. In human skin, electrical stimulation provoked a large flare reaction and at a frequency of 4 Hz both CGRP and SP concentrations increased significantly. In spite of the large flare reactions no protein extravasation was induced, which suggests major species differences. It will be of interest to investigate whether the lack of neurogenic protein extravasation is also valid under pathophysiological conditions.

Figures

Figure 1. Schematic diagram of the experimental set-up

Note that two or three microdialysis fibres were inserted in parallel to increase relative recovery of the microdialysate.

Figure 2. Time courses of CGRP concentration in the dialysate of intradermal microdialysis fibres after stimulating (filled bar) at 4 Hz (n = 17 rats; n = 7 humans) and 1 Hz (n = 15 rats; n = 5 humans) in comparison to the control values in rat and human skin

Because of the lower flow rate (1.6 and 2.5 μl min−1) CGRP levels at the 4 Hz stimulation are significantly higher for both species (P < 0.05). In rat (A), CGRP increase following the 4 Hz stimulation significantly exceeded that following the stimulation at 1 Hz and outlasted the stimulation period. In human skin (B), the stimulation at 1 Hz did not induce any increase in CGRP, whereas the stimulation at 4 Hz provoked a significant release of CGRP, which remained at a higher level during the whole observation time. Control values remained at a constant baseline level. At both frequencies CGRP levels in rat skin are significantly above those in human skin (P < 0.05). (***P < 0.001, **P < 0.01 and *P < 0.05; Scheffé‘s post hoc test.)

Figure 3. Time courses of substance P in rat (1 Hz, n = 15; 4 Hz, n = 13) and human (1 Hz, n = 5; 4 Hz, n = 7) skin

In rat skin significantly higher SP levels could be measured than in human skin (P < 0.05). According to the lower flow rate neuropeptide concentrations in the 4 Hz protocol are significantly higher compared with the 1 Hz protocol in both species (P < 0.001). In rat skin (A), a significant release of SP was observed during stimulation at 1 Hz, whereas the increase at the higher stimulation frequency did not reach a significant level. In contrast, in human skin (B) the stimulation at 1 Hz did not show any effects, whereas with the elevated stimulation frequency a significant SP release could be detected. Control values remained at an almost constant baseline level in both protocols. (**P < 0.01 and *P < 0.05; ANOVA, Scheffé‘s post hoc test.)

Figure 4. Protein extravasation in electrically stimulated and non-stimulated capillaries in rats (1 Hz, n = 15; 4 Hz, n = 17) and humans (1 Hz, n = 5; 4 Hz, n = 8)

In rat skin (A), total protein content exponentially declined after insertion of the microdialysis fibres to a baseline level, from which electrical stimulation evoked a significant increase of protein at both 1 and 4 Hz. In contrast, protein content in the dialysate derived from the non-stimulated capillaries continued to decrease gradually. Following 4 Hz stimulation protein extravasation was elevated during the complete sampling period, whereas at the 1 Hz stimulation only the first washout value was significantly increased. In human skin (B), plasma extravasation exponentially decreased in the stimulated and non-stimulated capillaries at both frequencies. In both species the 4 Hz protein values were significantly elevated compared with the 1 Hz values due to the lower perfusion rate (rat skin, P < 0.001; human skin, P < 0.05). (***P < 0.001, **P < 0.01 and *P < 0.05, ANOVA, Scheffé‘s post hoc test.)