Comparison of electrically induced flare response patterns in human and pig skin.

OBJECTIVE: We compared the characteristics of neurogenic flare responses in human and pig skin to establish a translational research animal model. MATERIAL AND SUBJECTS: Eight domestic pigs and six male subjects were investigated. TREATMENT: Electrical pulses were delivered transcutaneously with increasing current intensities, pulse frequencies and pulse widths. METHODS: Inflammatory skin responses were recorded by laser Doppler imaging and analyzed by ANOVA and Fisher's (LSD) post hoc test. RESULTS: Transcutaneous stimuli of 5 mA onward induced a significant flare development in humans. In the pig, significantly lower currents of 2.5 mA already induced a flare response. Smaller flare sizes of about 3.5 cm(2) were analyzed. The flare continuously declined despite ongoing stimulation. CONCLUSIONS: Lower excitation thresholds and smaller receptive fields of nociceptors can be suggested in pigs. Impaired neuropeptide release, altered vesicle replenishment, different neuropeptide sensitivity, or insufficient peripheral decoding of action potentials may contribute to steadily decreasing flare responses. These attributes may be objectives of pre-clinical anti-hyperalgesic studies and their accurate analysis in pigs reveals a particularly sensitive translational animal model for nociceptor researches.

Fast modulation of heat-activated ionic current by proinflammatory interleukin 6 in rat sensory neurons.

The pro-inflammatory cytokine interleukin-6 (IL-6) together with its soluble receptor (sIL-6R) induces and maintains thermal hyperalgesia. It facilitates the heat-induced release of calcitonin gene-related peptide from rat cutaneous nociceptors in vivo and in vitro. Here we report that exposure of nociceptive neurons to the IL-6-sIL-6R complex or the gp130-stimulating designer IL-6-sIL-6R fusion protein Hyper-IL-6 (HIL-6) resulted in a potentiation of heat-activated inward currents (I(heat)) and a shift of activation thresholds towards lower temperatures without affecting intracellular calcium levels. The Janus tyrosine kinase inhibitor AG490, the selective protein kinase C (PKC) inhibitor, bisindolylmaleimide 1 (BIM1), as well as rottlerin, a selective blocker of the PKCdelta isoform, but not the cyclooxygenase inhibitor indomethacin, effectively reduced the effect. Reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed expression of mRNA for the signal-transducing beta subunit of the receptor gp130 in neuronal somata, rather than satellite cells in rat dorsal root ganglia. Together, the results suggest that IL-6-sIL-6R acts directly on sensory neurons. It increases their susceptibility to noxious heat via the gp130/Jak/PKCdelta signalling pathway.

Differential time course of NGF-induced hyperalgesia to heat versus mechanical and electrical stimulation in human skin.

BACKGROUND: Nerve growth factor (NGF) causes early heat and delayed mechanical hyperalgesia. Axonal transport might contribute to lasting responses. Temporal hyperalgesia development was investigated by administering NGF in paraspinal skin. Transient receptor potential ankyrin 1 (TRPA1) is up-regulated by NGF and chemical responsiveness to cinnamon aldehyde (TRPA1 agonist) was quantified. METHODS: Eight healthy volunteers received 1 µg human recombinant NGF (i.d. 50 µL) to L4/L5 processi spinosi skin. Mechanical, thermal and electrical sensitization was assessed at 3-6 h and at days 1, 2, 3, 5, 7, 10, 14 and 21, and pain upon cinnamon aldehyde (20%, 60 µL) recorded at days 3 and 21. RESULTS: Heat hyperalgesia developed with an initial maximum at 3 h [heat pain threshold -3.9°; peak pain ratings +22 visual analogue scale (VAS)] that decreased by day 1, subsequently increased to a maximum around day 5 (-5 ± 0.2 °C, +41 ± 4 VAS), and thereafter declined to ∼20% at day 21. Mechanical and electrical hyperexcitability developed within 3 days and gradually increased to peak between days 14 and 21. Pain intensity upon cinnamon aldehyde stimulation was doubled at the NGF site at day 3 and was still increased by about 50% at day 21. CONCLUSIONS: NGF causes immediate heat hyperalgesia probably linked to an up-regulation and sensitization of transient receptor potential vanilloid 1 and possibly other proteins involved in heat transduction. The delayed mechanical hyperalgesia is apparently independent of the time required for axonal transport of NGF receptor complexes. Local mRNA translation at axonal terminals and protein accumulation is hypothesized being involved in sustained NGF-evoked hyperalgesia.

The inflammatory mediators serotonin, prostaglandin E2 and bradykinin evoke calcium influx in rat sensory neurons.

The inflammatory mediators bradykinin, prostaglandin E(2) and serotonin interact to excite and sensitize nociceptive neurons. All three mediators are coupled to signaling pathways that potentially induce rises in intracellular calcium concentration in other models. The aim of this study was therefore to investigate if the three mediators cause calcium rises in isolated rat sensory neurons that may explain their sensitizing action. Neurons exposed to serotonin, bradykinin, and prostaglandin E(2) exhibited reversible increases in intracellular calcium concentration, which were absent in calcium-free solution. The calcium increase induced by serotonin was preserved in the presence of extracellular cadmium suggesting calcium influx potentially through the serotonin receptor ion channel 5-HT(3). The bradykinin-induced calcium response was slower, showed pronounced tachyphylaxis and was absent in the presence of extracellular cadmium ions. Similar results were obtained for prostaglandin E(2) although the calcium rises were fast and not prone to tachyphylaxis. This suggests that prostaglandin E(2) as well as bradykinin via activation of G protein-coupled receptors seem to couple to calcium-permeant ion channels possibly the heat-transducing vanilloid receptor type 1 or related ion channels. The three mediators, however, did not cooperate to induce supra-additive calcium responses when applied simultaneously. In summary, our results suggest that the inflammatory mediators serotonin, prostaglandin E(2) and bradykinin induce calcium influx in sensory neurons. However, they do not utilize a calcium-dependent cooperative mechanism to facilitate proton-induced currents.

Axonal hyperexcitability after combined NGF sensitization and UV-B inflammation in humans.

BACKGROUND: Both nerve growth factor (NGF) and ultraviolet-B (UV-B) irradiation sensitize nociceptive nerve endings and increase axonal excitability of nociceptors. Combining NGF and UV-B treatment is supra-additive for sensory sensitization and even caused spontaneous pain in about 70% of the subjects. METHODS: UV-B irradiation was performed at day 21 after intradermal NGF injection in 13 volunteers. Pain thresholds, electrically induced axon reflex erythema and pain (1.5-fold pain threshold, 5-100 Hz) was analysed at days 22, 24, 28, 35, 49 and 70 and correlated to hyperalgesia and spontaneous pain. RESULTS: Electrical pain threshold after combined NGF/UVB was reduced below single treatment at 24 h but not at 72 h post-UV-B irradiation. At the NGF/UV-B site, electrical pain was enhanced at all frequencies compared with single NGF and UV-B sites at 24 and 72 h with pain ratings exceeding control values about twofold to threefold [65 ± 7 vs. 25 ± 8 visual analogue scale (VAS) (24 h) and 55 ± 9 vs. 22 ± 5 VAS (72 h)]. Hyperalgesia to electrical stimulation correlated with hyperalgesia to pinprick (Spearman r = 0.44; p < 0.001, Bonferroni corr.) and supra-threshold heat (Spearman r = 0.55; p < 0.001) stimulation at 24 h only. Electrical pain thresholds at the NGF/UV-B site weakly correlated to spontaneous pain levels (Spearman r = 0.3; p = 0.025, without Bonferroni correction). In contrast, electrically induced pain or axon reflex erythema did not correlate to spontaneous pain levels. CONCLUSIONS: The combination of NGF and UV-B increases axonal excitability that contributes to hyperalgesia and might also facilitate ongoing spontaneous pain.

Differential effects of lidocaine on nerve growth factor (NGF)-evoked heat- and mechanical hyperalgesia in humans.

We investigated the effects of a non-specific sodium channel blocker (lidocaine) on heat pain thresholds and mechanical impact pain at day 7 and 21 after intradermal injection of 1 µg NGF. Measurements were performed in 12 healthy male subjects prior to and 5 min after intradermal injection of 150 µl lidocaine administered at concentrations of 0.01% (∼0.4 mM) and 0.1% (∼4 mM) to both NGF and control skin sites. NGF caused a maximum reduction of heat pain thresholds at day 7 (NGF 42.6 ± 0.6 vs. 49.4 ± 0.3 °C in control skin). Lidocaine sensitized normal skin for heat pain, but reduced heat hyperalgesia after NGF at day 7 (44.3 ± 0.8 °C, lidocaine 0.1%; p < 0.005). Pain upon supra-threshold mechanical impact stimulation was increased after NGF at day 7 (VAS 29 + 5) and massively enhanced at day 21 (VAS 64 + 5, p < 0.001). Lidocaine dose-dependently attenuated mechanically-induced pain at both control and NGF-treated sites. Maximum lidocaine effects on mechanical hyperalgesia were recorded at day 21 in NGF skin (pain reduction to VAS 37 ± 4, p < 0.00001). Repetitive impact stimuli caused increasingly more pain at the NGF sites at day 21 and this pain increase was efficiently suppressed by lidocaine 0.1%. Lidocaine differentially affects NGF-induced mechanical hyperalgesia (analgesic effect) and heat sensitivity of nociceptors (sensitizing effect). These opposing responses may be attributed to block of sodium channels vs. sensitization of TRPV1. NGF-evoked extreme mechanical impact pain indicates high action potential discharge frequencies, which might be more susceptible to lidocaine block.

Fast Ca2+-induced potentiation of heat-activated ionic currents requires cAMP/PKA signaling and functional AKAP anchoring.

Calcium influx and the resulting increase in intracellular calcium concentration ([Ca(2+)](i)) can induce enhanced sensitivity to temperature increases in nociceptive neurons. This sensitization accounts for heat hyperalgesia that is regularly observed following the activation of excitatory inward currents by pain-producing mediators. Here we show that rat sensory neurons express calcium-dependent adenylyl cyclases (AC) using RT-PCR and nonradioactive in situ hybridization. Ionomycin-induced rises in [Ca(2+)](i)-activated calcium-dependent AC and caused translocation of catalytic protein kinase A subunit. Elevation of [Ca(2+)](i) finally resulted in a significant potentiation of heat-activated currents and a drop in heat threshold. This was not prevented in the presence of suramin that nonspecifically uncouples G protein-dependent receptors. The sensitization was, however, inhibited when the specific PKA antagonist PKI(14-22) was added to the pipette solution or when PKA coupling to A kinase anchoring protein (AKAP) was disrupted with InCELLect StHt-31 uncoupling peptide. The results show that heat sensitization in nociceptive neurons can be induced by increases in [Ca(2+)](i) and requires PKA that is functionally coupled to the heat transducer, mostly likely vanilloid receptor VR-1. This calcium-dependent pathway can account for the sensitizing properties of many excitatory mediators that activate cationic membrane currents.