Roman Rukwied’s research while affiliated with Heidelberg University and other places

Introduction Injection of recombinant human nerve growth factor (rhNGF) evokes acute heat and prolonged “polymodal” (mechanosensitive [CM]) and “silent” (mechano in sensitive [CMi]) C-nociceptor sensitization. Both nociceptor classes can be activated differentially using slowly depolarizing electrical sinusoidal stimuli. Objectives To explore the temporal profile of nociceptor sensitization to heat and mechanical and electrical stimuli in humans after rhNGF. Methods Recombinant human nerve growth factor (1 µg) and NaCl (0.9%) was injected into human forearm skin (n = 9, 50 µL/injection). Pain ratings (numeric rating scale) to transcutaneous electrical stimuli (1 ms 20 Hz rectangular pulses, 500-ms half-period sine wave [1 Hz] and 4 Hz sine wave pulses [2.5 and 60 seconds]) were assessed at days 3, 21, and 49 after injection, in addition to heat pain thresholds (HPTs, 9 × 9 mm thermode) and mechanical impact pain (4 and 8 m/second). Results Suprathreshold sinusoidal stimulation for specific CM (1 Hz) and combined CM and CMi (4 Hz) activation resulted in enhanced pain from day 3 post rhNGF and lasted throughout 7 weeks. These temporal dynamics contrasted minimum HPTs at day 3 (normalized by day 49) or mechanical impact pain (developing slowly until day 21 before declining depending on stimulus intensity). Correlation analyses of electrical pain indicated diverging kinetics when assessed for CM with or without concomitant CMi activation at days 3 and 21, which converged 7 weeks post rhNGF. Conclusions Exceptionally long sensitization of CM and CMi nociceptors by rhNGF, uncovered by suprathreshold electrical sinusoidal stimulation, indicates a signal transduction–independent long-lasting hyperexcitability of C-nociceptors that clinically may contribute to rhNGF-maintained chronic inflammatory pain.

Background We examined de‐functionalization and temporal functional recovery of C‐nociceptor evoked pain after topical 8% capsaicin applied for 4 consecutive days. Methods Capsaicin and placebo patches were applied to human forearm skin ( n = 14). Cold, warmth and heat pain thresholds, pain NRS to electrical and thermal (48°C, 5 s) stimuli and axon reflex flare were recorded weekly for 49 days. Mechanical and heat sensitive (‘polymodal’) nociceptors were activated by single electrical half‐period sinusoidal pulses (0.5 s, 1 Hz). Mechanical and heat in sensitive (‘silent’) nociceptors were activated by 4 Hz sinusoidal stimuli. Results Capsaicin abolished heat pain. Sensation to electrical sinusoidal stimulation was reduced but never abolished during the treatment. Pain to electrical 1 Hz ‘polymodal’ nociceptor stimulation took longer to recover than pain ratings to 4 Hz 2.5 s sinusoidal stimulation activating ‘polymodal’ and ‘silent’ nociceptors (35 vs. 21 days). Heat pain was indifferent to placebo from day 21–49. Axon reflex flare was abolished during capsaicin and only recovered to ~50% even after 49 days. Conclusions Capsaicin abolishes heat transduction at terminal nociceptive endings, whereas small‐diameter axons sensitive to sinusoidal electrical stimulation can still be activated. 1 Hz depolarizing stimuli evoke burst discharges, as demonstrated before, and recover slower after capsaicin than single pulses induced by 4 Hz. The difference in recovery suggests differential time course of functional regeneration for C‐nociceptor sub‐types after capsaicin. All sensations recovered completely within 7 weeks in healthy subjects. Our findings contrast analgesia lasting for months in spontaneous neuropathic pain patients treated with 8% capsaicin. Significance Sinusoidal electrical stimulation can still activate small diameter axons desensitized to heat after 4 consecutive days of topical 8% capsaicin application and reveals differential temporal functional regeneration of C‐nociceptor sub‐types. Electrical sinusoidal stimulation may detect such axons that no longer respond to heat stimuli in neuropathic skin.

The majority of somatosensory DRG neurons express GABAA receptors (GABAAR) and depolarise in response to its activation based on the high intracellular chloride concentration maintained by the Na-K-Cl cotransporter type 1 (NKCC1). The translation of this response to peripheral nerve terminals in people is so far unclear. We show here that GABA (EC50 = 16.67μM) acting via GABAAR produces an influx of extracellular calcium in approximately 20% (336/1720) of isolated mouse DRG neurons. In contrast, upon injection into forearm skin of healthy volunteers GABA (1mM, 100μl) did not induce any overt sensations nor a specific flare response and did not sensitize C-nociceptors to slow depolarizing electrical sinusoidal stimuli. Block of the inward chloride transporter NKCC1 by furosemide (1mg/100μl) did not reduce electrically evoked pain ratings nor did repetitive GABA stimulation in combination with an inhibited NKCC1 driven chloride replenishment by furosemide. Finally, we generated a sustained period of C-fiber firing by iontophoretically delivering codeine or histamine to induce tonic itch. Neither the intensity nor the duration of histamine or codeine itch was affected by prior injection of furosemide. We conclude that although GABA can evoke calcium transients in a proportion of isolated mouse DRG neurons, it does not induce or modify pain or itch ratings in healthy human skin even when chloride gradients are altered by inhibition of the sodium coupled NKCC1 transporter.

Introduction & Objective: We aimed to assess C-fiber excitability via transcutaneous-electrical nerve stimulation (TENS) in diabetic sensorimotor neuropathy (DSPN) and its association with epidermal innervation and quantitative sensory testing. Pain perception was evaluated in glucose-tolerant humans (n=14) and patients with type 2 diabetes: no-DSPN (n=15), possible-DSPN (n=21), probable-DSPN (n=19), and confirmed-DSPN (n=11). Subsequent analysis compared patients with positive (n=17) and negative symptoms (n=16). Methods: Slow depolarizing transcutaneous currents of low-intensity with 4 Hz sinusoidal stimulation profile and single 500 ms half sine wave pulses were used to stimulate C-fibers. Pain perception quantified using (0-10)-Numeric-Rating-Scale, detection/pain thresholds and pain-change-dynamics were assessed. Results: Confirmed-DSPN had diminished pain perception (1.72±0.50 vs 4.98±0.46), elevated detection (0.19±0.04 vs 0.05±0.01) and pain thresholds (0.28±0.04 vs 0.1±0.01) and reduced pain habituation compared to the control group. Elevated pain perception (pos:7.32±0.72 vs neg:3.81±0.98) and lowered detection/pain thresholds (pos:0.24±0.04 vs neg:0.13±0.03) were shown in the positive symptoms group compared to the negative symptoms group (p<0.05). Over 50% of patients with DSPN experience elevated pain with electrical stimuli in regions with reduced heat pain sensitivity, while less than 6% show similar responses in areas with diminished mechanical pain sensitivity indicating axonal degeneration. Conclusions: Our results suggest that DSPN proceeds via differential transductional disruption towards denervation. TENS results, influenced by positive symptoms, confirm C-fibers' role for the clinical burden of DSPN. TENS offers further insights into DSPN's natural course, aiding high-risk patient identification and guiding interventions. Disclosure O. Eldesouky: None. L. Seebauer: Other Relationship; Springer Medizin Verlag GmbH. R. Rukwied: None. R. Carr: None. M. Roshan: None. A. Sulaj: None. D. Tsilingiris: None. S. Kopf: Speaker's Bureau; Lilly Diabetes, Bayer Inc. T.H. Fleming: None. M. Schmelz: Consultant; Lilly GmbH, Merz Therapeutics, Bayer Inc., Medtronic GmbH. J. Szendroedi: None. Z. Kender: None.

Aim. Evaluating pain sensation after C nociceptor activation with transdermal sinusoidal current wave stimulation protocol and skin biopsy. Materials and methods. Healthy volunteers aged 20–30 years (17 females and 18 males) participated after having given their informed consent. Half‑sine wave pulses of 0.5 sec duration (1 Hz) were generated at intensities of 0.2 to 1 mA by a constant current stimulator. Apart from half‑sine wave stimulation, sine wave pulses of 60 sec duration (4 Hz) were generated at intensity of 0.2 mA also by the constant current stimulator (Digitimer Ltd, Welwyn Garden City, UK) controlled by DAPSYS 8 (www.dapsys.net). Moreover, we performed 3‑mm skin punch biopsies 10 cm above the lateral malleolus of the leg and in the middle of the volar side of the forearm to the volunteer’s group. Results. Delivering transdermal sinusoidal half‑sine wave when trying to stimulate mechano‑sensitive C fibers, when the amplitude of the delivered wave is increased from 0.2 to 1 mA according to our protocol, pain sensation is also increased following the same scheme. If we observe a different scheme of activation in C fibers, this could be a sign of neuropathic pain. Considering the mechano‑insensitive C fibers of pain, when trying to stimulate them we expect increasing pain sensation and then familiarization, desensitization and reduction of pain sensation. As a result, if this scheme isn’t observed when sine wave is delivered transdermal with 1 min of duration, and we observe a different scheme, a C fiber neuropathy and neuropathic pain could be involved. Regarding the skin biopsies, a correlation between pain sensation of sine wave (delivered transdermal to stimulate mechano‑insensitive C fibers of pain on the forearm), and the nerve fiber density was observed. A correlation between the bifurcated fibers of the biopsy site and the pain sensation was observed when mechanosensitive and mechano‑insensitive fibers are stimulated, which needs further investigation. Also, a correlation between the remnant nerve fibers of subepidermal nerve plexus and mechano‑insensitive nerve fibers of pain is observed that also needs further investigation. Conclusion. Skin biopsy and transdermal electrical stimulation are very promising available tools of diagnosing C fiber neuropathies and assessing neuropathic pain.

Low-frequency sinusoidal current applied to human skin evokes local axon reflex flare and burning pain, indicative of C-fibre activation. Because topical cooling works well as a local analgesic, we examined the effect of cooling on human pain ratings to sinusoidal and rectangular profiles of constant current stimulation. Unexpectedly, pain ratings increased upon cooling the skin from 32 to 18°C. To explore this paradoxical observation, the effects of cooling on C-fibre responses to stimulation with sinusoidal and rectangular current profiles were determined in ex vivo segments of mouse sural and pig saphenous nerve. As expected by thermodynamics, the absolute value of electrical charge required to activate C-fibre axons increased with cooling from 32°C to 20°C, irrespective of the stimulus profile used. However, for sinusoidal stimulus profiles, cooling enabled a more effective integration of low-intensity currents over tens of milliseconds resulting in a delayed initiation of action potentials. Our findings indicate that the paradoxical cooling-induced enhancement of electrically evoked pain in people can be explained by an enhancement of C-fibre responsiveness to slow depolarization at lower temperatures. This property may contribute to symptoms of enhanced cold sensitivity, especially cold allodynia, associated with many forms of neuropathic pain.