Otilia Obreja

IL-1β potentiates heat-activated currents in rat sensory neurons: involvement of IL-1RI, tyrosine kinase, and protein kinase C

Interleukin 1β (IL‐1β) is a proinflammatory cytokine that maintains thermal hyperalgesia and facilitates the release of calcitonin gene‐related peptide from rat cutaneous nociceptors in vivo and in vitro. Brief applications of IL‐1β to nociceptive neurons yielded a potentiation of heat‐activated inward currents (Iheat) and a shift of activation threshold toward lower temperature without altering intracellular calcium levels. The IL‐1β‐induced heat sensitization was not dependent on G‐protein‐coupled receptors but was mediated by activation of protein kinases. The nonspecific protein kinase inhibitor staurosporine, the specific protein kinase C inhibitor bisindolylmaleimide BIM1, and the protein tyrosine kinase inhibitor genistein reduced the sensitizing effect of IL‐1β whereas negative controls were ineffective. RT‐PCR and in situ hybridization revealed IL‐1RI but not RII expression in neurons rather than surrounding satellite cells in rat dorsal root ganglia. IL‐1β acts on sensory neurons to increase their susceptibility for noxious heat via an IL‐1RI/PTK/ PKC‐dependent mechanism.—Obreja, O., Rathee, P. K., Lips, K. S., Distler, C., Kress, M. IL‐1J potentiates heat‐activated currents in rat sensory neurons: involvement of IL‐1RI, tyrosine kinase, and protein kinase C. FASEB J. 16, 1497–1503 (2002)

NGF induces non-inflammatory localized and lasting mechanical and thermal hypersensitivity in human skin

&NA; Nerve growth factor (NGF) modulates sensitivity and sprouting of nociceptors. We explored the spatial and temporal sensitization induced by NGF injection (1 &mgr;g) 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 °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.

Interleukin-6 in combination with its soluble IL-6 receptor sensitises rat skin nociceptors to heat, in vivo

&NA; 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°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.

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.

Patterns of activity-dependent conduction velocity changes differentiate classes of unmyelinated mechano-insensitive afferents including cold nociceptors, in pig and in human

&NA; Activity‐dependent slowing of conduction velocity (ADS) differs between classes of human nociceptors. These differences likely reflect particular expression and use‐dependent slow inactivation of axonal ion channels and other mechanisms governing axonal excitability. In this study, we compared ADS of porcine and human cutaneous C‐fibers. Extracellular recordings were performed from peripheral nerves, using teased fiber technique in pigs and microneurography in humans. We assessed electrically‐induced conduction changes and responsiveness to natural stimuli. In both species, the group of mechano‐insensitive C‐fibers showed the largest conduction slowing (˜30%) upon electrical stimulation (2 Hz for 3 min). In addition, we found mechano‐insensitive cold nociceptors in pig that slowed only minimally (<10% at 2 Hz), and a similar slowing pattern was found in some human C‐fibers. Mechano‐sensitive afferents showed an intermediate conduction slowing upon 2 Hz stimulation (pig: 14%, human 23%), whereas sympathetic efferent fibers in pig and human slowed only minimally (5% and 9%, respectively). In fiber classes with more pronounced slowing, conduction latencies recovered slower; i.e. mechano‐insensitive afferents recovered the slowest, followed by mechano‐sensitive afferents whereas cold nociceptors and sympathetic efferents recovered the fastest. We conclude that mechano‐insensitive C‐fiber nociceptors can be differentiated by their characteristic pattern of ADS which are alike in pig and human. Notably, cold nociceptors with a distinct ADS pattern were first detected in pig. Our results therefore suggest that the pig is a suitable model to study nociceptor class‐specific changes of ADS.

Modeling activity-dependent changes of axonal spike conduction in primary afferent C-nociceptors

Action potential initiation and conduction along peripheral axons is a dynamic process that displays pronounced activity dependence. In patients with neuropathic pain, differences in the modulation of axonal conduction velocity by activity suggest that this property may provide insight into some of the pathomechanisms. To date, direct recordings of axonal membrane potential have been hampered by the small diameter of the fibers. We have therefore adopted an alternative approach to examine the basis of activity-dependent changes in axonal conduction by constructing a comprehensive mathematical model of human cutaneous C-fibers. Our model reproduced axonal spike propagation at a velocity of 0.69 m/s commensurate with recordings from human C-nociceptors. Activity-dependent slowing (ADS) of axonal propagation velocity was adequately simulated by the model. Interestingly, the property most readily associated with ADS was an increase in the concentration of intra-axonal sodium. This affected the driving potential of sodium currents, thereby producing latency changes comparable to those observed for experimental ADS. The model also adequately reproduced post-action potential excitability changes (i.e., recovery cycles) observed in vivo. We performed a series of control experiments replicating blockade of particular ion channels as well as changing temperature and extracellular ion concentrations. In the absence of direct experimental approaches, the model allows specific hypotheses to be formulated regarding the mechanisms underlying activity-dependent changes in C-fiber conduction. Because ADS might functionally act as a negative feedback to limit trains of nociceptor activity, we envisage that identifying its mechanisms may also direct efforts aimed at alleviating neuronal hyperexcitability in pain patients.

Neurogenic components of trypsin- and thrombin-induced inflammation in rat skin, in vivo

Abstract:  Activation of protease‐activated receptors (PAR) can induce vasodilation (VD) and increase of vascular permeability either directly by stimulating endothelial cells or indirectly via activation of nociceptors and subsequent release of neuropeptides (neurogenic inflammation). We aimed to estimate the relative contribution of the two pathways for stimulation with endogenous activators of PAR‐2 (trypsin) and of PAR‐1, 3 and 4 (thrombin) using in vivo dermal microdialysis in rats. Protein extravasation (PE) was assessed by increase of protein concentration in the dialysate, and VD was quantified by laser Doppler scanning. Both trypsin (10−8−10−4 M) and thrombin (10−6, 10−5.5 and 10−5 M) provoked PE and local VD in a dose‐dependent manner. Trypsin (10−4 M)‐induced PE was inhibited by 87.2 ± 21% due to the substance P (SP) NK1 receptor antagonist SR140333. VD was blocked by 58.15 ± 10.1% in response to the calcitonin gene‐related peptide (CGRP) receptor antagonist CGRP8‐37. By contrast, CGRP8‐37 did not affect thrombin‐induced VD, while blockade of SP receptors prevented the PE elicited only by low doses of thrombin (10−6 M), being ineffective at higher thrombin concentrations. In conclusion, intradermal trypsin elicits a neurogenic inflammation in rat, probably mediated via PAR‐2 activation on nociceptors and subsequent SP and CGRP release. Thrombin‐induced PE and VD are mediated mainly by a non‐neurogenic mechanism.

NGF enhances electrically induced pain, but not axon reflex sweating.

&NA; 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/m2, 20 Hz: 9 mL/h/m2, 100 Hz: 10 mL/h/m2). 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. NGF administration increased electrically induced pain, decreased activity‐dependent slowing of mechano‐insensitive nociceptors, but did not affect sympathetic efferents. Axonal sensitization could underlie the NGF‐evoked hyperalgesia.

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.

Nerve growth factor selectively decreases activity-dependent conduction slowing in mechano-insensitive C-nociceptors

Summary Intracutaneous nerve growth factor reduces the activity‐dependent slowing of conduction of the mechano‐insensitive C‐fibers. This long‐term change of axonal characteristics might contribute to hyperalgesia. Parts of this study have been published in abstract form (Obreja et al., Society for Neuroscience Meeting, 2008, Washington, 668.5). ABSTRACT 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.