Sensitization of supra-threshold pain responses-Translational aspects and mechanisms.

A substantial translational gap in pain research has been reflected by a mismatch of relevant primary pain assessment endpoints in preclinical vs. clinical trials. Since activity-dependent mechanisms may be neglected during reflexive tests, this may add as a confounding factor during preclinical pain assessment. In this perspective, we consider the evidence for a need for supra-threshold pain assessment in the pain research literature. In addition to that, we focus on previous results that may demonstrate an example mechanism, where the detection of neuron-glial interactions on pain seems to be substantially depending on the assessment of pain intensity beyond threshold levels.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

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.

Sympathetic efferent neurons are less sensitive than nociceptors to 4 Hz sinusoidal stimulation.

BACKGROUND: Sinusoidal current stimuli preferentially activate C-nociceptors. Sodium channel isoforms NaV1.7 and NaV1.8 have been implicated in this. Sympathetic efferent neurons lack NaV1.8 and were explored upon sinusoidal activation. METHODS: Quantitative Sudomotor Axon Reflex Test (QSART) was performed in hairy (n = 16) and glabrous (n = 12) skin. Responses of sympathetic efferents (n = 10) and nociceptive afferents (n = 21) to sinusoidal current stimulation (4 Hz, 0.05-0.15 mA) were recorded in humans by microneurography (n = 11). Activation of sympathetic units upon supra-threshold sinusoidal currents (>0.8 mA) was recorded in pigs (n = 8). RESULTS: Sinusoidal stimuli (4 Hz, 0.4 mA) evoked weak sweat output (30 ml/h/m2 ) in hairy skin compared to rectangular pulses (4 Hz, 5 mA, 53 ml/h/m2 , p < .00001, ANOVA). No change in sweat output was recorded from glabrous skin to sine wave stimuli. Sinusoidal current at intensities ranging from 0.05 to 0.15 mA activated almost all (85%) nociceptors but only 40% of sympathetic units in human. Stimuli lead to a significantly lower activation in sympathetic versus nociceptive fibres as measured by activity-dependent slowing (ADS) of conduction (sympathetic efferents average ADS 100 ± 0.2% vs. C-nociceptors average ADS 113 ± 4%, p < .003, ANOVA). CONCLUSIONS: Sympathetic efferent neurons are less apt to convert slow depolarizations into action potentials as compared to nociceptors. Distinctive sodium channel expression patterns between nociceptors and sympathetic efferent neurons may account for this difference. Sinusoidal stimulation therefore provokes weak sweat responses and provides no alternative for clinical assessment of autonomic function. SIGNIFICANCE: C-nociceptors in hairy skin are activated by 4 Hz sinusoidal current stimulation at lower intensities than myelinated fibres. Sympathetic efferent neurons-albeit also unmyelinated-are less responsive to sinusoidal activation than nociceptors within the same skin area. Cutaneous sympathetic efferent neurons apparently are less apt than nociceptors to convert slow depolarization into action potentials.

Tuning in C-nociceptors to reveal mechanisms in chronic neuropathic pain.

OBJECTIVE: Develop and validate a low-intensity sinusoidal electrical stimulation paradigm to preferentially activate C-fibers in human skin. METHODS: Sinusoidal transcutaneous stimulation (4Hz) was assessed psychophysically in healthy volunteers (n = 14) and neuropathic pain patients (n = 9). Pursuing laser Doppler imaging and single nociceptor recordings in vivo in humans (microneurography) and pigs confirmed the activation of "silent" C-nociceptors. Synchronized C-fiber compound action potentials were evoked in isolated human nerve fascicles in vitro. Live cell imaging of L4 dorsal root ganglia in anesthetized mice verified the recruitment of small-diameter neurons during transcutaneous 4-Hz stimulation of the hindpaw (0.4mA). RESULTS: Transcutaneous sinusoidal current (0.05-0.4mA, 4Hz) activated "polymodal" C-fibers (50% at ∼0.03mA) and "silent" nociceptors (50% at ∼0.04mA), intensities substantially lower than that required with transcutaneous 1-ms rectangular pulses ("polymodal" ∼3mA, "silent" ∼50mA). The stimulation induced delayed burning (nonpulsating) pain and a pronounced axon-reflex erythema, both indicative of C-nociceptor activation. Pain ratings to repetitive stimulation (1 minute, 4Hz) adapted in healthy volunteers by Numeric Rating Scale (NRS) -3 and nonpainful skin sites of neuropathic pain patients by NRS -0.5, whereas pain even increased in painful neuropathic skin by approximately NRS +2. INTERPRETATION: Sinusoidal electrical stimulation at 4Hz enables preferential activation of C-nociceptors in pig and human skin that accommodates during ongoing (1-minute) stimulation. Absence of such accommodation in neuropathic pain patients suggest axonal hyperexcitability that could be predictive of alterations in peripheral nociceptor encoding and offer a potential therapeutic entry point for topical analgesic treatment. Ann Neurol 2018;83:945-957.

Activation of glial glutamate transporter via MAPK p38 prevents enhanced and long-lasting non-evoked resting pain after surgical incision in rats.

Pain after surgery has recently become a major issue not only due to lack of treatment success in the acute phase; even more alarming is the large number of patients developing prolonged pain after surgery. Because spinal glutamate as well as spinal glia plays a major role in acute incisional pain, we investigated the role of the spinal glial glutamate transporters (GT), GLAST, GLT-1, for acute and prolonged pain and hyperalgesia caused by an incision. Spinal administration of the GT-inhibitor DL-TBOA increased non-evoked pain but not evoked pain behavior (hyperalgesia) up to 2 weeks after incision. In accordance, spinal GLAST (and to a lesser degree GLT-1) were upregulated after incision for several days. Long-term incision induced GT upregulation was prevented by long-lasting p38-inhibitor administration but not by long-lasting ERK1/2-inhibition after incision. In accordance, daily treatment with the p38-inhibitor (but not the ERK1/2 inhibitor) prolonged non-evoked but not evoked pain behavior after incision. In electrophysiological experiments, spontaneous activity of high threshold (HT) (but not wide dynamic range (WDR)) neurons known to transmit incision induced non-evoked pain was increased after prolonged treatment with the p38-inhibitor. In conclusion, our findings indicate a new spinal pathway by which non-evoked pain behavior after incision is modulated. The pathway is modality (non-evoked pain) and neuron (HT) specific and disturbance contributes to prolonged long-term pain after surgical incision. This may have therapeutic implications for the treatment of acute and - even more relevant - for prevention of chronic pain after surgery in patients.

Assessment of TTX-s and TTX-r Action Potential Conduction along Neurites of NGF and GDNF Cultured Porcine DRG Somata.

Nine isoforms of voltage-gated sodium channels (NaV) have been characterized and in excitable tissues they are responsible for the initiation and conduction of action potentials. For primary afferent neurons residing in dorsal root ganglia (DRG), individual neurons may express multiple NaV isoforms extending the neuron's functional capabilities. Since expression of NaV isoforms can be differentially regulated by neurotrophic factors we have examined the functional consequences of exposure to either nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) on action potential conduction in outgrowing cultured porcine neurites of DRG neurons. Calcium signals were recorded using the exogenous intensity based calcium indicator Fluo-8®, AM. In 94 neurons, calcium signals were conducted along neurites in response to electrical stimulation of the soma. At an image acquisition rate of 25 Hz it was possible to discern calcium transients in response to individual electrical stimuli. The peak amplitude of electrically-evoked calcium signals was limited by the ability of the neuron to follow the stimulus frequency. The stimulus frequency required to evoke a half-maximal calcium response was approximately 3 Hz at room temperature. In 13 of 14 (93%) NGF-responsive neurites, TTX-r NaV isoforms alone were sufficient to support propagated signals. In contrast, calcium signals mediated by TTX-r NaVs were evident in only 4 of 11 (36%) neurites from somata cultured in GDNF. This establishes a basis for assessing action potential signaling using calcium imaging techniques in individual cultured neurites and suggests that, in the pig, afferent nociceptor classes relying on the functional properties of TTX-r NaV isoforms, such as cold-nociceptors, most probably derive from NGF-responsive DRG neurons.