Benjamin Weinkauf

NGF-evoked sensitization of muscle fascia nociceptors in humans.

TOC summary Prolonged local hyperalgesia after nerve growth factor injection in muscle fascia suggests that sensitization of fascia nociceptors contributes to clinical muscle pain. Abstract 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 pH 4 (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.

NGF Sensitizes Nociceptors for Cowhage- but Not Histamine-Induced Itch in Human Skin

TO THE EDITOR Chronic itch has been associated with increased levels of epidermal nerve growth factor (NGF) and enhanced neuronal signaling, for instance in atopic dermatitis (AD) patients (Tominaga et al., 2009; Yamaguchi et al., 2009). Experimentally, pruritus can be elicited in AD independent of histamine (Ikoma et al., 2004). Spicules from Mucuna pruriens (cowhage) also produce sensations of itch when inserted into the epidermis of humans (Namer et al., 2008; Sikand et al., 2009). Cowhage activates mechanoresponsive ‘‘polymodal’’ C-nociceptors in humans and also Ad fibers in monkey (Ringkamp et al., 2011) and evokes itch sensations at an intensity comparable to histamine but without an accompanying axon reflex flare reaction. Our understanding of nonhistaminergic itch has increased tremendously during the past decade. Increased levels of NGF mRNA and elevated keratinocyte and serum NGF content have both been linked to itch in animal models (Takano et al., 2005; Yamaura et al., 2011). Anti-NGF treatment in these animals significantly reduced dermatitis scores, the number of epidermal nerve fibers, and scratching behavior (Takano et al., 2005). In AD patients, enhanced levels of NGF in epidermal and horny layers correlated with the severity of itching (Tominaga et al., 2009; Yamaguchi et al., 2009). We therefore hypothesized that NGF sensitizes skin nociceptors in AD patients and thereby increases pruritus. In healthy volunteers, intradermally administered NGF chronically sensitizes nociceptors, resulting in a localized hyperalgesia to heat and a prolonged mechanical hyperalgesia (Rukwied et al., 2010; Obreja et al., 2011). It is unclear whether such NGF-mediated nociceptor sensitization also modulates the sensitivity to various pruritic stimuli. We therefore injected 1 mg NGF intradermally in healthy male volunteers (n1⁄413, age 36±11) and investigated their itch response to histamine iontophoresis (15 mC) and cowhage spicule insertion (15–20 spicules). Stimuli were applied at a time point coinciding with the development of maximum heat (day 7) and maximum mechanical hyperalgesia (day 21) determined in a previous human trial (Rukwied et al., 2010). Assuming that histamineand cowhage-induced itch are mediated by separate classes of primary afferent neurons (Johanek et al., 2007; Namer et al., 2008), our expectation was that the time course of heat hyperalgesia would parallel that of increased histamine sensitivity (mediated by mechano-insensitive ‘‘silent’’ nociceptors) following NGF and similarly that the time courses of mechanical hyperalgesia and cowhage-induced itch (mediated by ‘‘polymodal’’ nociceptors) would be comparable (Namer et al., 2008). Maximum histamine-induced itch within the NGF-treated site did not differ between day 7 (28±5, visual analog scale (VAS)) and day 21 (VAS 27±7). VAS area under the curve (AUC) values calculated during the 10-min recording period were also similar at days 7 (492±122) and 21 (489±112). There were no significant differences between histamine-induced itch responses recorded from treated and untreated skin (Po0.6, Wilcoxon test, Figure 1a). Epidermal application of cowhage spicules at the NGF-treated site on day 7 evoked a peak itch sensation of VAS 25±5 and an AUC of 359±69, both of which were not significantly different from values obtained from control skin (VAS 23±6, AUC 349±73). In contrast, 21 days after NGF treatment, cowhageinduced itch was significantly enhanced compared with the control sites, with a maximum itch of VAS 35±7 (Po0.05, Wilcoxon test) at day 21 and a corresponding AUC of 522±98 (Po0.02, Wilcoxon test, Figure 1b). To differentially quantify ‘‘silent’’ nociceptor sensitization, we also analyzed the axon reflex flare using laser Doppler imaging. In comparison with control skin, NGF did not augment histamine-induced flare responses at either day 7 (16±1 vs. 18±2 cm) or day 21 (15±2 vs. 16±1 cm). As in humans (Johanek et al., 2007), no axon reflex flare was apparent after application of cowhage, neither at the control nor at the NGF site (flare area o0.8±0.2 cm, data not shown). We further assessed heat pain thresholds and mechanical pain for pressure (100 kPa), pinprick (150 mN), and impact stimuli (8 m s) (Rukwied et al., 2010). Heat pain thresholds after NGF were reduced by 5±0.4 1C at day 7 and 21, but this reduction did not correlate with the histamineor enhanced cowhage-induced itch (Spearman’s r1⁄4 0.059, P1⁄4 0.77 (histamine) and r1⁄4 0.023, P1⁄4 0.91 (cowhage), Figure 2a). In contrast, pain upon mechanical stimulation increased after NGF (VAS from 3±0.8 to 19±4 (pressure), VAS from 3±1 to 9±3 (pinprick), and VAS from 5±2 to 21±3 (impact)) and each of these increases correlated with cowhage-induced itch sensations (Spearman’s r1⁄4 0.67, Po0.0002 (pressure), r1⁄4 0.7, Po0.00001 (pinprick), r1⁄40.55, Po0.003 (impact pain, Figure 2b)). LETTERS TO THE EDITOR

Nerve growth factor induces sensitization of nociceptors without evidence for increased intraepidermal nerve fiber density

Summary Three weeks after nerve growth factor injection in pigs we found nociceptor sensitization possibly underlying NGF‐evoked hyperalgesia in human skin without evidence for nociceptor sprouting. 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 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 mm2. 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.

Local Gene Expression Changes after UV-Irradiation of Human Skin

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.

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.

Inflammation meets sensitization--an explanation for spontaneous nociceptor activity?

Summary Experimental combination of nerve growth factor (NGF) evoked sensitization and ultraviolet‐B (UV‐B)–induced inflammation causes spontaneous pain and supra‐additive hyperalgesia in human beings. The data reinforce therapeutic analgesic anti‐NGF strategies. Abstract Anti‐nerve growth factor (anti‐NGF) treatment is analgesic in chronic inflammatory pain conditions without reducing inflammation. Hypothesizing that ongoing pain induced by inflammatory mediators is increased by long term sensitization of nociceptors, we combined the non‐inflammatory NGF‐sensitization model with an inflammatory ultraviolet‐B (UV‐B) model in human volunteers. UV‐B irradiation of the skin presensitized with NGF 3 weeks before intensified the pre‐existing NGF hyperalgesia during the inflammatory phase of UV‐B and caused spontaneous pain in about 70% of the subjects. Pain levels paralleled the intensity of UVB inflammation. Hyperalgesia recorded on a VAS (0–100) was additive after combined NGF/UV‐B treatment versus single NGF or UV‐B treatment for mechanical impact and tonic heat stimuli, again paralleling the intensity of the UV‐B inflammation. In contrast, ratings to tonic mechanical pressure (100 kPa for 10 seconds, peak VAS 58 ± 7 vs VAS 21 ± 5 [NGF] and VAS 12 ± 3 [UV‐B]) and pinprick (150 mN for 5 seconds, peak VAS 33 ± 7 vs VAS 10 ± 2 [NGF] and VAS 8 ± 3 [UV‐B]) increased in a supra‐additive manner. This supra‐additive effect faded 24 hours after irradiation, although heat sensitization remained increased. Hyperalgesia and spontaneous pain coexisted in NGF/UV‐B treated skin but did not significantly correlate (r < −0.1 at day 1 and r < 0.2 at day 3). We conclude that NGF can sensitize nociceptive endings such that inflammatory mediators may cause sufficient excitation to provoke spontaneous pain. Our results suggest that neuronal sensitization and level of inflammation represent independent therapeutic targets in chronic inflammatory pain conditions.

Comparison of nerve growth factor–induced sensitization pattern in lumbar and tibial muscle and fascia

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

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

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.

Mechano-sensitive nociceptors are required to detect heat pain thresholds and cowhage itch in human skin

Mechano‐sensitive and mechano‐insensitive C‐nociceptors in human skin differ in receptive field sizes and electrical excitation thresholds, but their distinct functional roles are yet unclear.

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

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.