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Introduction: Nerve growth factor (NGF) induces profound hyperalgesia. In this study we explored patterns of NGF sensitization in muscle and fascia of distal and paraspinal sites., Methods: We injected 1 µg of NGF into human (n = 8) tibialis anterior and erector spinae muscles and their fasciae. The spatial extent of pressure sensitization, pressure pain threshold, and mechanical hyperalgesia (150 kPa, 10 s) was assessed at days 0.25, 1, 3, 7, 14, and 21. Chemical sensitization was explored by acidic buffer injections (pH 4, 100 µl) at days 7 and 14., Results: The mechanical hyperalgesia area was larger in tibial fascia than in muscle. Pressure pain thresholds were lower, tonic pressure pain ratings, and citrate buffer evoked pain higher in fascia than in muscle., Conclusions: Spatial mechanical sensitization differs between muscle and fascia. Thoracolumbar fasciae appear more sensitive than tibial fasciae and may be major contributors to low back pain, but the temporal sensitization profile is similar between paraspinal and distal sites. Muscle Nerve 52: 265-272, 2015., (© 2014 Wiley Periodicals, Inc.)

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., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Nerve growth factor (NGF) modulates sensitivity and sprouting of nociceptors. We explored the spatial and temporal sensitization induced by NGF injection (1 microg) in human skin. Hyperalgesia was investigated in 16 volunteers (36+/-9 years) at day 1, 3, 7, 21, and 49. Areas of mechanical (brush, pin-prick) and heat (43 degrees C) sensitization were mapped and thermal (heat and cold) pain thresholds, mechanical (impact stimulation) and electrically evoked pain, and axon reflex flare were assessed. No spontaneous pain or local inflammation was recorded upon NGF injection and during 49 days. Sensitization to heat was maximum at day 3 and lasted 21 days. Hyperalgesia to cold was recorded at day 7 and 21. Hypersensitivity to mechanical impact stimuli developed delayed, reached maximum at day 21, and persisted throughout 49 days. Fifty percent of all volunteers reported a static allodynia to tonic pressure until day 21. Electrical stimulation at 7.5 mA was more painful at the NGF site at day 21, which correlated significantly to maximum impact pain. Axon reflex flare was unaffected by NGF. Sensitization was limited to the NGF injection site, no touch- or pin-prick evoked secondary hyperalgesia was observed. Spatially restricted hyperalgesia indicates a peripheral rather than central mechanism. The temporal profile of lasting nociceptor sensitization suggests an altered peripheral axonal expression of sensory proteins specifically leading to mechanical and thermal sensitization. Intradermal NGF administration provokes a pattern of sensitization that can be used as experimental model for neuropathic pain.

Inflammatory mediators not only activate "pain-"sensing neurons, the nociceptors, to trigger acute pain sensations, more important, they increase nociceptor responsiveness to produce inflammatory hyperalgesia. For example, prostaglandins activate G(s)-protein-coupled receptors and initiate cAMP- and protein kinase A (PKA)-mediated processes. We demonstrate for the first time at the cellular level that heat-activated ionic currents were potentiated after exposure to the cAMP activator forskolin in rat nociceptive neurons. The potentiation was prevented in the presence of the selective PKA inhibitor PKI(14-22), suggesting PKA-mediated phosphorylation of the heat transducer protein. PKA regulatory subunits were found in close vicinity to the plasma membrane in these neurons, and PKA catalytic subunits only translocated to the cell periphery when activated. The translocation and the current potentiation were abolished in the presence of an A-kinase anchoring protein (AKAP) inhibitor. Similar current changes after PKA activation were obtained from human embryonic kidney 293t cells transfected with the wild-type heat transducer protein vanilloid receptor 1 (VR-1). The forskolin-induced current potentiation was greatly reduced in cells transfected with VR-1 mutants carrying point mutations at the predicted PKA phosphorylation sites. The heat transducer VR-1 is therefore suggested as the molecular target of PKA phosphorylation, and potentiation of current responses to heat depends on phosphorylation at predicted PKA consensus sites. Thus, the PKA/AKAP/VR-1 module presents as the molecular correlate of G(s)-mediated inflammatory hyperalgesia.

Interleukin-6 (IL-6) contributes to increased pain and hyperalgesia in inflamed tissue. We have investigated the effects of IL-6, alone or in combination with its soluble receptor (sIL-6R), on the sensitivity of nociceptors to noxious heat, using dermal microdialysis. Plasmapheresis membranes were inserted into the abdominal skin of adult male Wistar rats (n=46) and perfused with modified Ringer solution. After three control samples (20 min each), the skin area above the membrane was heated to 48 degrees C for 20 min. The stimulation was followed by two washout samples. The calcitonin gene-related peptide (CGRP) content of the dialysate was measured with an enzyme immunoassay. Heat stimulation provoked a significant CGRP increase in the dialysate. Intradermal application of IL-6 (200 ng ml-1) did not significantly alter heat-induced CGRP release. However, a significant sensitisation of the heat-induced CGRP release was observed when sIL-6R (25 ng ml-1) was applied, either alone or in combination with IL-6. Neutralisation of endogenous IL-6 with a sheep anti-rat IL-6 serum did not alter heat-induced CGRP release, but abolished the sIL-6R-mediated sensitising effect. We show that IL-6 in combination with its soluble receptor can sensitise nociceptors to heat and provide evidence for the constitutive expression of the signalling molecule gp130, but not of the IL-6-membrane-bound (specific) receptor, in nociceptors.

Nerve growth factor (NGF) injected into the human skin causes local hyperalgesia to mechanical and electrical stimuli lasting for weeks. Pig data suggested axonal sensitization of C-nociceptors as a contributing mechanism. Here, we recorded single C-nociceptors in 11 human subjects 3 weeks after intracutaneous injection of 1 μg NGF into the foot dorsum. For each identified unit, the receptive field was mapped and, whenever possible, we recorded 2 terminal branches of the same unit, 1 from the hyperalgesic NGF-site ("inside") and the other from the nonsensitized skin ("outside"). In the saline-treated control feet, mechano-insensitive nociceptors (CMi) were more abundant than at the NGF sites (36% vs 19%). Units with axonal properties of CMi fibres but displaying positive mechanical responses ("CMi-like") dominated at the NGF site (27% vs 6%). Moreover, axonal branches innervating the hyperalgesic skin displayed significantly lower electrical thresholds and less activity-dependent conduction velocity slowing when compared with "outside" or control skin. The "inside" branches also showed long-lasting after-discharges and less adaptation to repeated mechanical stimuli. NGF-induced long-term nociceptor hyperexcitability was maximum at the terminal branches directly treated with NGF. The sensitization included sensory and axonal components affecting both activation thresholds and supra-threshold responses. Our data suggest that a combination of sensory sensitization and axonal hyperexcitability is underlying the localized hyperalgesia by facilitating action potential generation and conduction. Axonal changes were also found in the asymptomatic skin surrounding the NGF-treatment sites, thereby possibly reflecting "nociceptive priming." [ABSTRACT FROM AUTHOR]

Abstract: Inflammation and trauma lead to tissue acidification and release of inflammatory mediators, including prostaglandin E2 (PGE2). Protons can evoke pain through acid-sensing ion channels (ASICs) and TRPV1 receptors. In this study, we examined whether PGE2 can potentiate proton-induced nociception in humans on injection into skin and muscle. Psychophysical and vascular responses to microinjections of protons (pH 6.0 and 6.5), PGE2 (10-6 and 10-7 M) and their combinations into forearm skin (30 μL) or anterior tibial muscle (50 μL) were assessed in 16 male subjects. Pain intensity, axon reflex erythema, and heat pain thresholds were recorded after skin challenge; pain intensity and thresholds for pressure-evoked pain were recorded after intramuscular injections. Intradermal or intramuscular injections of PGE2 induced very low levels of pain similar to saline. Administration of low pH caused moderate pain within 5 seconds that declined rapidly over 15 to 20 seconds. In comparison, coinjection of low pH with PGE2 led to a biphasic profile of the pain response. Combined pH + PGE2 stimulation provoked significantly increased pain in the second phase after injections (20 to 100 seconds) both in skin and muscle, whereas the initial injection pain was not enhanced. Heat pain thresholds were reduced after PGE2 and combined pH + PGE2, whereas flare responses were rather attenuated on coadministration of low pH with PGE2. Intriguingly, when compared with skin, muscle pain was significantly lower in the initial phase (0 to 15 seconds) but significantly higher in the second phase (20 to 100 seconds after injection). Perspective: PGE2 can potentiate nociceptor activation by protons in human skin and muscle, indicated by increased sustained pain ratings. This can be best explained by TRPV1 sensitization in the presence of PGE2, a mechanism potentially relevant for inflammatory and injury-induced pain. [Copyright &y& Elsevier]

Abstract: Ultraviolet B (UVB) irradiation induces hyperalgesia in human and animal pain models. We investigated mechanical sensitization, increase in axonal excitability, and spontaneous activity in different C-nociceptor classes after UVB in pig skin. We focused on units with receptive fields covering both irradiated and nonirradiated skin allowing intraindividual comparisons. Thirty-five pigs were irradiated in a chessboard pattern, and extracellular single-fibre recordings were obtained 10 to 28 hours later (152 fibers). Units from the contralateral hind limb served as a control (n = 112). Irradiated and nonirradiated parts of the same innervation territory were compared in 36 neurons; low threshold C-touch fibers (n = 10) and sympathetic efferents (n = 2) were unchanged, but lower mechanical thresholds and higher discharge frequency at threshold were found in mechanosensitive nociceptors (n = 12). Half of them could be activated with nonnoxious brush stimuli in the sunburn. Four of 12 mechanoinsensitive nociceptors were found sensitized to mechanical stimulation in the irradiated part of the receptive field. Activity-dependent slowing of conduction was reduced in the irradiated and in the nonirradiated skin as compared with the control leg, whereas increased ability to follow high stimulation frequencies was restricted to the sunburn (108.5 ± 37 Hz UVB vs 6.3 ± 1 Hz control). Spontaneous activity was more frequent in the sunburn (72/152 vs 31/112). Mechanical sensitization of primary nociceptors and higher maximum after frequency are suggested to contribute to primary hyperalgesia, whereas the spontaneous activity of silent nociceptors might offer a mechanistic link contributing to ongoing pain and facilitated induction of spinal sensitization. [ABSTRACT FROM AUTHOR]

Abstract: 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 3weeks 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 20mN. After NGF application, the mechanical receptive fields of nociceptors increased from 25 to 43mm2. 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. [Copyright &y& Elsevier]