Nerve growth factor (NGF) is involved in the long-term sensitization of nociceptive processing linked to chronic pain. Functional and structural ("sprouting") changes can contribute. Thus, humans report long-lasting hyperalgesia to mechanical and electrical stimulation after intradermal NGF injection and NGF-induced sprouting has been reported to underlie cancer bone pain and visceral pain. Using a human-like animal model we investigated the relationship between the structure and function of unmyelinated porcine nociceptors 3 weeks after intradermal NGF treatment. Axonal and sensory characteristics were studied by in vivo single-fiber electrophysiology and immunohistochemistry. C fibers recorded extracellularly were classified based on mechanical response and activity-dependent slowing (ADS) of conduction velocity. Intraepidermal nerve fiber (IENF) densities were assessed by immunohistochemistry in pigs and in human volunteers using the same NGF model. NGF increased conduction velocity and reduced ADS and propagation failure in mechano-insensitive nociceptors. The proportion of mechano-sensitive C nociceptors within NGF-treated skin areas increased from 45.1% (control) to 71% and their median mechanical thresholds decreased from 40 to 20 mN. After NGF application, the mechanical receptive fields of nociceptors increased from 25 to 43 mm(2). At the structural level, however, IENF density was not increased by NGF. In conclusion, intradermal NGF induces long-lasting axonal and mechanical sensitization in porcine C nociceptors that corresponds to hyperalgesia observed in humans. Sensitization is not accompanied by increased IENF density, suggesting that NGF-induced hyperalgesia might not depend on changes in nerve fiber density but could be linked to the recruitment of previously silent nociceptors.

Nerve growth factor (NGF) induces acute sensitization of nociceptive sensory endings and long-lasting hyperalgesia. NGF modulation of sodium channel expression might contribute to neurotrophin-induced hyperalgesia. Here, we investigated NGF-evoked changes of the activity-dependent slowing of conduction in porcine C-fibers. Animals received intradermal injections of NGF (2 μg or 8 μg) or saline in both hind limbs. Extracellular recordings from the saphenous nerves were performed 1 week later. Based on sensory thresholds and electrically induced activity-dependent slowing (ADS) of axonal conduction, C-fibers were classified as mechano-sensitive afferents, mechano-insensitive afferents, cold nociceptors, and sympathetic efferents. NGF (2 μg) increased conduction velocity in C-fibers from 1.0±0.05 m/s to 1.2±0.07 m/s. In mechano-insensitive afferents, NGF (8 μg) reduced activity-dependent slowing of conduction, from 5.3±0.2% to 3.2±0.5% (0.125-0.5 Hz stimulation) and from 28.5±1.3% to 20.9±1.9% (2 Hz stimulation), such that ADS no longer differentiated between mechano-sensitive and mechano-insensitive fibers. Accordingly, the number of fibers with pronounced ADS decreased but more units with pronounced ADS were mechano-sensitive. Spontaneously active C-fibers were increased above the control level (1%) by NGF 8 μg (8%). The results demonstrate that NGF changes the functional axonal characteristics of mechano-insensitive C-fibers and enhances spontaneous activity thereby possibly contributing to hyperalgesia.

Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

Single intradermal injections of nerve growth factor (NGF) evoke prolonged but temporally distinct sensitization patterns to somatosensory stimuli. Focal administration of the non-histaminergic pruritogen cowhage but not histamine resulted in elevated itch at day 21 after NGF administration. Here, we injected bovine adrenal medulla peptide 8-22 (BAM8-22), β-alanine (β-ALA) and endothelin-1 (ET-1) into NGF-treated skin of 11 healthy volunteers and investigated the corresponding itch/pain and flare reactions. β-ALA was the weakest pruritogen, while BAM8-22 and ET-1 were equally potent as histamine. NGF did not sensitize itch or flare reactions induced by any compound, but injection and evoked pain were increased at day 21 and 49. The involvement of histamine H1 receptors in itch was explored in eight subjects after oral cetirizine. ET-1-induced itch and flare were significantly reduced. BAM8-22 and β-ALA itch were not affected, but flare responses after BAM8-22 reduced by 50%. The results indicate that a single NGF injection does not sensitize for experimentally induced itch but increases pain upon pruritogen injection. In healthy humans, pruritic and algetic processing appear differentially regulated by NGF. However, in patients suffering chronic itch, prolonged elevation of NGF-levels under inflammatory conditions may contribute to elevated itch.

Nerve growth factor (NGF) induces local hyperalgesia for a few days after intramuscular injection, but longer-lasting muscle pain upon systemic administration. As the muscle fascia is densely innervated by free nerve endings, we hypothesized a lasting sensitization of fascia nociceptors by NGF. We administered 1 μg NGF (dissolved in 100 μL saline) ultrasound-guided to the fascia of the Musculus erector spinae muscle at the lumbar level of 14 male volunteers and assessed hypersensitivity after 6 hours, and 1, 3, 7, 14, and 21 days. Pain upon mechanical stimuli (constant pressure and dynamic impact), upon exercise and electrically induced M. erector spinae contraction, and upon injection of 100 μL phosphate buffer pH4 (at day 7 and 14 only) to the fascia of both NGF- and saline-treated muscles, was investigated. Injections into the muscle fascia did not cause acute pain. Local heat pain thresholds were unchanged following NGF and saline (control) administration. NGF evoked a lasting (days 1-7) and significant reduction of pressure pain, pressure thresholds, exercise-evoked muscle pain, and hyperalgesia to impact stimuli (12 m/s). Pain upon injected protons was significantly elevated (P<0.04) for 2 weeks. NGF induced a sensitization of the muscle fascia to mechanical and chemical stimuli lasting for up to 2 weeks. As nociceptors in the fascia appear to be particularly prone to sensitization, they may contribute to acute or chronic muscle pain.

High-affinity receptors for nerve growth factor (NGF) are found on nociceptors and sympathetic efferents. NGF is known to sensitize nociceptors, increase innervation density, and fire frequency of sympathetic fibers. We explored axonal sensitization of afferent and efferent fibers following intracutaneous injection of NGF in human and pig skin. In humans, frequency-dependent (5, 20, 100 Hz) electrically induced pain was assessed 1, 3, 7, 21, and 49 days post injection. Sweat output was recorded in parallel using the quantitative sudomotor axon reflex test (QSART). Electrically induced pain ratings (7.5 mA for 30 s) significantly increased at the NGF sites for 5 Hz (numeric rating scale [NRS] 6±0.5 vs 3.7±0.4), 20 Hz (NRS 7.2±0.4 vs 5±0.5), and 100 Hz stimulation (NRS 6.9±0.4 vs 5.4±0.3) at day 21, and also for 5 Hz at day 49 (NRS 5.4±0.4 vs 3.8±0.3). Electrically evoked QSART increased frequency dependent, but was not altered by NGF throughout the entire observation period (average QSART at 5 Hz: 3 mL/h/m(2), 20 Hz: 9 mL/h/m(2), 100 Hz: 10 mL/h/m(2)). Similarly, NGF did not change the activity-dependent slowing of conduction of sympathetic efferents (6±2% vs 5.1±1.5%, for 3 minutes, 2 Hz) in pig single-fiber recordings. In parallel to the increased pain ratings recorded in humans, activity-dependent slowing of mechano-insensitive nociceptors was reduced by NGF (18.1±2% vs 29±1.4%). In summary, axonal sensitization of nociceptors by NGF could underlie the hyperalgesia to electrical stimulation. Enhanced responses were limited to nociceptors, as no sensitization was found in sympathetic efferent neurons.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, "silent" nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.