Timothy V. Hartke

A Role for Nociceptive, Myelinated Nerve Fibers in Itch Sensation

Despite its clinical importance, the underlying neural mechanisms of itch sensation are poorly understood. In many diseases, pruritus is not effectively treated with antihistamines, indicating the involvement of nonhistaminergic mechanisms. To investigate the role of small myelinated afferents in nonhistaminergic itch, we tested, in psychophysical studies in humans, the effect of a differential nerve block on itch produced by intradermal insertion of spicules from the pods of a cowhage plant (Mucuna pruriens). Electrophysiological experiments in anesthetized monkey were used to investigate the responsiveness of cutaneous, nociceptive, myelinated afferents to different chemical stimuli (cowhage spicules, histamine, capsaicin). Our results provide several lines of evidence for an important role of myelinated fibers in cowhage-induced itch: (1) a selective conduction block in myelinated fibers substantially reduces itch in a subgroup of subjects with A-fiber-dominated itch, (2) the time course of itch sensation differs between subjects with A-fiber- versus C-fiber-dominated itch, (3) cowhage activates a subpopulation of myelinated and unmyelinated afferents in monkey, (4) the time course of the response to cowhage is different in myelinated and unmyelinated fibers, (5) the time of peak itch sensation for subjects with A-fiber-dominated itch matches the time for peak response in myelinated fibers, and (6) the time for peak itch sensation for subjects with C-fiber-dominated itch matches the time for the peak response in unmyelinated fibers. These findings demonstrate that activity in nociceptive, myelinated afferents contributes to cowhage-induced sensations, and that nonhistaminergic itch is mediated through activity in both unmyelinated and myelinated afferents.

Peripherally acting mu-opioid receptor agonist attenuates neuropathic pain in rats after L5 spinal nerve injury.

&NA; Studies in experimental models and controlled patient trials indicate that opioids are effective in managing neuropathic pain. However, side effects secondary to their central nervous system actions present barriers to their clinical use. Therefore, we examined whether activation of the peripheral mu‐opioid receptors (MORs) could effectively alleviate neuropathic pain in rats after L5 spinal nerve ligation (SNL). Systemic loperamide hydrochloride (0.3–10 mg/kg, s.c.), a peripherally acting MOR‐preferring agonist, dose‐dependently reversed the mechanical allodynia at day 7 post‐SNL. This anti‐allodynic effect produced by systemic loperamide (1.5 mg/kg, s.c.) was blocked by systemic pretreatment with either naloxone hydrochloride (10 mg/kg, i.p.) or methyl‐naltrexone (5 mg/kg, i.p.), a peripherally acting MOR‐preferring antagonist. It was also blocked by ipsilateral intraplantar pretreatment with methyl‐naltrexone (43.5 μg/50 μl) and the highly selective MOR antagonist CTAP (5.5 μg/50 μl). However, this anti‐allodynic effect of systemic loperamide was not blocked by intraplantar pretreatment with the delta‐opioid receptor antagonist naltrindole hydrochloride (45.1 μg/50 μl). The anti‐allodynic potency of systemic loperamide varied with time after nerve injury, with similar potency at days 7, 28, and 42 post‐SNL, but reduced potency at day 14 post‐SNL. Ipsilateral intraplantar injection of loperamide also dose‐dependently (10–100 μg/50 μl) reversed mechanical allodynia on day 7 post‐SNL. We suggest that loperamide can effectively attenuate neuropathic pain, primarily through activation of peripheral MORs in local tissue. Therefore, peripherally acting MOR agonists may represent a promising therapeutic approach for alleviating neuropathic pain.

Activity-dependent slowing of conduction velocity in uninjured L4 C fibers increases after an L5 spinal nerve injury in the rat

Abstract Growing evidence suggests that uninjured afferents may play an important role in neuropathic pain following nerve injury. The excitability of nociceptive neurons in the L4 spinal nerve appears to be enhanced following an injury to the adjacent L5 spinal nerve. In this study, we investigated whether the action‐potential conduction properties of unlesioned, unmyelinated fibers are also altered. A teased‐fiber technique was used to record from single C fibers from the L4 spinal nerve of the rat in vitro. Repeated electrical stimulation of the tibial nerve was used to investigate activity‐dependent slowing of conduction velocity. Twin pulse stimulation at a 50 ms interpulse interval allowed investigation of supranormal conduction velocity. Blinded experiments were performed 8–10 days after sham surgery and after an L5 spinal nerve ligation (L5 SNL). Activity‐dependent slowing revealed two populations of C fibers, a “nociceptor” population with a large degree of activity‐dependent slowing and a “non‐nociceptor” population with a smaller degree of activity‐dependent slowing. Both populations showed enhanced activity‐dependent slowing of conduction velocity and enhanced supranormal conduction velocities in lesioned animals compared to sham animals. Activity‐dependent slowing was also enhanced after an L5 SNL in the mouse. These alterations in conduction velocity may reflect changes in expression of ion channels responsible for the membrane excitability. These data provide additional evidence that a nerve injury leads to persistent alterations in the properties of adjacent uninjured, unmyelinated fibers.

Three functionally distinct classes of C-fibre nociceptors in primates

In primate C-fiber polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically-sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of β-alanine, a MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to β-alanine, and their response is only half that of QCs. Mechanically-insensitive C-fibers (C-MIAs) are β-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that β-alanine and histamine activate distinct populations of capsaicin responsive neurons in primate DRG. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with β-alanine responsive QC fibers likely corresponding to murine MrgprD- expressing, non-peptidergic nociceptive afferents.

Conduction Properties Distinguish Unmyelinated Sympathetic Efferent Fibers and Unmyelinated Primary Afferent Fibers in the Monkey

Background Different classes of unmyelinated nerve fibers appear to exhibit distinct conductive properties. We sought a criterion based on conduction properties for distinguishing sympathetic efferents and unmyelinated, primary afferents in peripheral nerves. Methodology/Principal Findings In anesthetized monkey, centrifugal or centripetal recordings were made from single unmyelinated nerve fibers in the peroneal or sural nerve, and electrical stimuli were applied to either the sciatic nerve or the cutaneous nerve endings, respectively. In centrifugal recordings, electrical stimulation at the sympathetic chain and dorsal root was used to determine the fibers origin. In centrifugal recordings, sympathetic fibers exhibited absolute speeding of conduction to a single pair of electrical stimuli separated by 50 ms; the second action potential was conducted faster (0.61 0.16%) than the first unconditioned action potential. This was never observed in primary afferents. Following 2 Hz stimulation (3 min), activity-dependent slowing of conduction in the sympathetics (8.6 0.5%) was greater than in one afferent group (6.7 0.5%) but substantially less than in a second afferent group (29.4 1.9%). In centripetal recordings, most mechanically-insensitive fibers also exhibited absolute speeding to twin pulse stimulation. The subset that did not show this absolute speeding was responsive to chemical stimuli (histamine, capsaicin) and likely consists of mechanically-insensitive afferents. During repetitive twin pulse stimulation, mechanosensitive afferents developed speeding, and speeding in sympathetic fibers increased. Conclusions/Significance The presence of absolute speeding provides a criterion by which sympathetic efferents can be differentiated from primary afferents. The differences in conduction properties between sympathetics and afferents likely reflect differential expression of voltage-sensitive ion channels.

Local loperamide injection reduces mechanosensitivity of rat cutaneous, nociceptive C-fibers.

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.

Sodium Channel Nav1.8 Underlies TTX-Resistant Axonal Action Potential Conduction in Somatosensory C-Fibers of Distal Cutaneous Nerves

Voltage-gated sodium (NaV) channels are responsible for the initiation and conduction of action potentials within primary afferents. The nine NaV channel isoforms recognized in mammals are often functionally divided into tetrodotoxin (TTX)-sensitive (TTX-s) channels (NaV1.1–NaV1.4, NaV1.6–NaV1.7) that are blocked by nanomolar concentrations and TTX-resistant (TTX-r) channels (NaV1.8 and NaV1.9) inhibited by millimolar concentrations, with NaV1.5 having an intermediate toxin sensitivity. For small-diameter primary afferent neurons, it is unclear to what extent different NaV channel isoforms are distributed along the peripheral and central branches of their bifurcated axons. To determine the relative contribution of TTX-s and TTX-r channels to action potential conduction in different axonal compartments, we investigated the effects of TTX on C-fiber-mediated compound action potentials (C-CAPs) of proximal and distal peripheral nerve segments and dorsal roots from mice and pigtail monkeys (Macaca nemestrina). In the dorsal roots and proximal peripheral nerves of mice and nonhuman primates, TTX reduced the C-CAP amplitude to 16% of the baseline. In contrast, >30% of the C-CAP was resistant to TTX in distal peripheral branches of monkeys and WT and NaV1.9−/− mice. In nerves from NaV1.8−/− mice, TTX-r C-CAPs could not be detected. These data indicate that NaV1.8 is the primary isoform underlying TTX-r conduction in distal axons of somatosensory C-fibers. Furthermore, there is a differential spatial distribution of NaV1.8 within C-fiber axons, being functionally more prominent in the most distal axons and terminal regions. The enrichment of NaV1.8 in distal axons may provide a useful target in the treatment of pain of peripheral origin. SIGNIFICANCE STATEMENT It is unclear whether individual sodium channel isoforms exert differential roles in action potential conduction along the axonal membrane of nociceptive, unmyelinated peripheral nerve fibers, but clarifying the role of sodium channel subtypes in different axonal segments may be useful for the development of novel analgesic strategies. Here, we provide evidence from mice and nonhuman primates that a substantial portion of the C-fiber compound action potential in distal peripheral nerves, but not proximal nerves or dorsal roots, is resistant to tetrodotoxin and that, in mice, this effect is mediated solely by voltage-gated sodium channel 1.8 (NaV1.8). The functional prominence of NaV1.8 within the axonal compartment immediately proximal to its termination may affect strategies targeting pain of peripheral origin.

Laser speckle imaging to improve clinical outcomes for patients with trigeminal neuralgia undergoing radiofrequency thermocoagulation

OBJECTIVE Percutaneous treatments for trigeminal neuralgia are safe, simple, and effective for achieving good pain control. Procedural risks could be minimized by using noninvasive imaging techniques to improve the placement of the radiofrequency thermocoagulation probe into the trigeminal ganglion. Positioning of a probe is crucial to maximize pain relief and to minimize unwanted side effects, such as denervation in unaffected areas. This investigation examined the use of laser speckle imaging during probe placement in an animal model. METHODS This preclinical safety study used nonhuman primates, Macaca nemestrina (pigtail monkeys), to examine whether real-time imaging of blood flow in the face during the positioning of a coagulation probe could monitor the location and guide the positioning of the probe within the trigeminal ganglion. RESULTS Data from 6 experiments in 3 pigtail monkeys support the hypothesis that laser imaging is safe and improves the accuracy of probe placement. CONCLUSIONS Noninvasive laser speckle imaging can be performed safely in nonhuman primates. Because improved probe placement may reduce morbidity associated with percutaneous rhizotomies, efficacy trials of laser speckle imaging should be conducted in humans.