Michael I. Nemenov

Ablation of rat TRPV1-expressing Adelta/C-fibers with resiniferatoxin: analysis of withdrawal behaviors, recovery of function and molecular correlates.

BackgroundAblation of TRPV1-expressing nociceptive fibers with the potent capsaicin analog resiniferatoxin (RTX) results in long lasting pain relief. RTX is particularly adaptable to focal application, and the induced chemical axonopathy leads to analgesia with a duration that is influenced by dose, route of administration, and the rate of fiber regeneration. TRPV1 is expressed in a subpopulation of unmyelinated C- and lightly myelinated Adelta fibers that detect changes in skin temperature at low and high rates of noxious heating, respectively. Here we investigate fiber-type specific behaviors, their time course of recovery and molecular correlates of axon damage and nociception using infrared laser stimuli following an RTX-induced peripheral axonopathy.ResultsRTX was injected into rat hind paws (mid-plantar) to produce thermal hypoalgesia. An infrared diode laser was used to stimulate Adelta fibers in the paw with a small-diameter (1.6 mm), high-energy, 100 msec pulse, or C-fibers with a wide-diameter (5 mm), long-duration, low-energy pulse. We monitored behavioral responses to indicate loss and regeneration of fibers. At the site of injection, responses to C-fiber stimuli were significantly attenuated for two weeks after 5 or 50 ng RTX. Responses to Adelta stimuli were significantly attenuated for two weeks at the highest intensity stimulus, and for 5 weeks to a less intense Adelta stimulus. Stimulation on the toe, a site distal to the injection, showed significant attenuation of Adelta responses for 7- 8 weeks after 5 ng, or 9-10 weeks after 50 ng RTX. In contrast, responses to C-fiber stimuli exhibited basically normal responses at 5 weeks after RTX. During the period of fiber loss and recovery, molecular markers for nerve regeneration (ATF3 and galanin) are upregulated in the dorsal root ganglia (DRG) when behavior is maximally attenuated, but markers of nociceptive activity (c-Fos in spinal cord and MCP-1 in DRG), although induced immediately after RTX treatment, returned to normal.ConclusionBehavioral recovery following peripheral RTX treatment is linked to regeneration of TRPV1-expressing Adelta and C-fibers and sustained expression of molecular markers. Infrared laser stimulation is a potentially valuable tool for evaluating the behavioral role of Adelta fibers in pain and pain control.

Inward currents in primary nociceptive neurons of the rat and pain sensations in humans elicited by infrared diode laser pulses.

&NA; Radiant heat is often used to study nociception in vivo. We now used infrared radiation generated by a diode laser stimulator (wavelength 980 nm) to investigate transduction mechanisms for noxious heat stimuli in acutely dissociated dorsal root ganglion (DRG) neurons of rats in vitro. The laser stimulator offered the unique opportunity to test whether the same stimuli also elicit pain sensations in humans. A specific heat‐induced current (Iheat) was elicited in six of 13 small DRG neurons (diameter ≤30 &mgr;m) tested in the whole‐cell configuration of the patch–clamp mode. Current responses in the seven heat‐insensitive neurons were within the range explainable by the temperature dependence of the recording setup. Iheat was characterized by: (1) non‐linearity of its amplitude during a suprathreshold heat ramp as well as with stimuli of increasing intensity with an estimated threshold of 42±1°C; (2) fast rise time and even faster decay time (t1/2=96.5±5.9 and 27.7±1.5 ms, respectively); and (3) rate dependence of its induction. All three heat‐sensitive neurons tested were also sensitive to capsaicin. The mean threshold for the induction of Iheat was 2.8±0.3 J mm−2. The threshold for the induction of action potentials by depolarizing current pulses was significantly reduced after laser stimulation, suggesting a sensitization at the transformation stage. No such change was seen in heat‐insensitive neurons that underwent the same heat stimuli. The same diode laser elicited pain sensations and laser‐evoked potentials in human subjects. No significant differences were seen between the pain thresholds in hairy and in glabrous skin, probably due to the deep penetration of this laser radiation. The mean pain threshold for stimuli ≥200 ms in humans was 2.5±0.2 J mm−2 (n=11), and did not differ from the thresholds for the induction of Iheat in vitro. Our results indicate that Iheat in primary sensory neurons can be activated by infrared laser pulses that are painful in humans.

Differential activation of trigeminal C or Aδ nociceptors by infrared diode laser in rats: behavioral evidence

Radiant heat is often used for studying thermal nociception, although inherent characteristics such as the broad spectrum of applied wavelengths of typical light sources limit control over and repeatability of stimuli. To overcome these problems, we used a diode infrared laser-based stimulator (wavelength: 980 nm) for selectively stimulating trigeminal Adelta or C thermonociceptors in rats. To provide indirect evidence for nociceptor-selective stimulation, we tested the effects of capsaicin, dimethylsulfoxide (DMSO), and morphine on withdrawal latencies for long pulses with a low current (hypothesized to selectively stimulate C nociceptors) and for threshold currents of short pulses with high current (hypothesized to selectively stimulate Adelta nociceptors) in lightly anesthetized rats. Nonmem analysis was used to perform pharmacodynamic modeling. The measured baseline withdrawal latency for long pulses was 12.5 +/- 0.3 s which was changed significantly to 6.7 +/- 0.4 s after applying topical capsaicin which selectively sensitizes C nociceptors and to 16.5 +/- 1.3 s after 1.0 mg/kg morphine which preferentially attenuates C fiber nociception. Topical DMSO which appears to selectively sensitize Adelta afferents did not significantly alter withdrawal latencies to the long pulses. Fitted threshold currents for short pulses after DMSO were however significantly lower (974 +/- 53 mA vs. 1113 +/- 12 mA for baseline) indicating Adelta sensitization. Capsaicin and morphine did not significantly change threshold currents. Best Nonmem fits for the long pulse were obtained using a model assuming no DMSO effect, but a different inter-individual variability after applying this substance. For the short pulse, a model assuming no capsaicin or morphine effect, but again allowing different inter-individual variabilities after applying these drugs, best described the data. We conclude that different settings of the stimulator used in this study were capable of selectively activating trigeminal Adelta or C thermonociceptors.

Differential brain activation associated with laser-evoked burning and pricking pain: An event-related fMRI study.

Abstract An important question remains as to how the brain differentially processes first (pricking) pain mediated by Aδ‐nociceptors versus second (burning) pain mediated by C‐nociceptors. In the present cross‐over randomized, within‐subjects controlled study, brain activity patterns were examined with event‐related fMRI while pricking and burning pain were selectively evoked using a diode laser. Stimuli evoking equivalent pain intensities were delivered to the dorsum of the left foot. Different laser parameters were used to elicit pricking (60 ms pulse duration) and burning (2.0 s pulse duration) pain. Whole brain group analysis showed that several brain areas were commonly activated by pricking and burning pain, including bilateral thalamus, bilateral anterior insula, bilateral posterior parietal lobule, contralateral dorsolateral prefrontal cortex, ipsilateral cerebellum, and mid anterior cingulate cortex. These findings show that pricking and burning pain were associated with activity in many of the same nociceptive processing brain regions. This may be expected given that Aδ‐and C‐nociceptive signals converge to a great extent at the level of the dorsal horn. Other brain regions showed differential processing. Stronger activation in the pricking pain condition was found in the ipsilateral hippocampus, bilateral parahippocampal gyrus, bilateral fusiform gyrus, contralateral cerebellum and contralateral cuneus/parieto‐occipital sulcus. Stronger activation in the burning pain condition was found in the ipsilateral dorsolateral prefrontal cortex. These differential activation patterns suggest preferential importance of Aδ‐fiber signals versus C‐fiber signals for these specific brain regions.

The modality‐specific contribution of peptidergic and non‐peptidergic nociceptors is manifest at the level of dorsal horn nociresponsive neurons

Ablation of TRPV1+/peptidergic or of MrgprD+/non‐peptidergic nociceptors produces modality‐specific deficits in the behavioural responses to heat and mechanical stimuli, respectively. Noxious heat‐induced dorsal horn Fos expression is also eliminated, despite the heat responsiveness of the non‐peptidergic nociceptors. To assess whether this modality‐specific contribution is manifest at the level of individual spinal neurons, we made extracellular recordings from mouse dorsal horn after selective ablation of the two nociceptor populations. Intrathecal capsaicin, which ablated the TRPV1+ nociceptors, abolished responsiveness of superficial and deep dorsal horn neurons to noxious heat, with no change in response to noxious mechanical stimulation. Ablation of MrgprD+ afferents did not alter the response to noxious heat but reduced the firing of dorsal horn neurons in response to noxious mechanical stimulation. These findings argue strongly that TRPV1+ and MrgprD+ nociceptors provide modality‐specific contributions to the response properties of spinal cord neurons.

Nociception and inflammatory hyperalgesia evaluated in rodents using infrared laser stimulation after Trpv1 gene knockout or resiniferatoxin lesion

Summary Chemoablation of TRPV1‐containing afferent fibers with resiniferatoxin produces more profound analgesia than TRPV1 receptor knockout assessed using either A&dgr;‐ or C‐fiber stimulation during baseline or inflamed conditions. ABSTRACT TRPV1 is expressed in a subpopulation of myelinated A&dgr; and unmyelinated C‐fibers. TRPV1+ fibers are essential for the transmission of nociceptive thermal stimuli and for the establishment and maintenance of inflammatory hyperalgesia. We have previously shown that high‐power, short‐duration pulses from an infrared diode laser are capable of predominantly activating cutaneous TRPV1+ A&dgr;‐fibers. Here we show that stimulating either subtype of TRPV1+ fiber in the paw during carrageenan‐induced inflammation or following hind‐paw incision elicits pronounced hyperalgesic responses, including prolonged paw guarding. The ultrapotent TRPV1 agonist resiniferatoxin (RTX) dose‐dependently deactivates TRPV1+ fibers and blocks thermal nociceptive responses in baseline or inflamed conditions. Injecting sufficient doses of RTX peripherally renders animals unresponsive to laser stimulation even at the point of acute thermal skin damage. In contrast, Trpv1−/− mice, which are generally unresponsive to noxious thermal stimuli at lower power settings, exhibit withdrawal responses and inflammation‐induced sensitization using high‐power, short duration A&dgr; stimuli. In rats, systemic morphine suppresses paw withdrawal, inflammatory guarding, and hyperalgesia in a dose‐dependent fashion using the same A&dgr; stimuli. The qualitative intensity of A&dgr; responses, the leftward shift of the stimulus‐response curve, the increased guarding behaviors during carrageenan inflammation or after incision, and the reduction of A&dgr; responses with morphine suggest multiple roles for TRPV1+ A&dgr; fibers in nociceptive processes and their modulation of pathological pain conditions.

Thermal nociceptive properties of trigeminal afferent neurons in rats

BackgroundAlthough nociceptive afferents innervating the body have been heavily studied form many years, much less attention has been paid to trigeminal afferent biology. In particular, very little is known concerning trigeminal nociceptor responses to heat, and almost nothing in the rat. This study uses a highly controlled and reproducible diode laser stimulator to investigate the activation of trigeminal afferents to noxious skin heating.ResultsThe results of this experiment demonstrate that trigeminal thermonociceptors are distinct from themonociceptors innervating the limbs. Trigeminal nociceptors have considerably slower action potential conduction velocities and lower temperature thresholds than somatic afferent neurons. On the other hand, nociceptors innervating both tissue areas separate into those that respond to short pulse, high rate skin heating and those that respond to long pulse, low rate skin heating.ConclusionsThis paper provides the first description in the literature of the in vivo properties of thermonociceptors in rats. These finding of two separate populations aligns with the separation between C and A-delta thermonociceptors innervating the paw, but have significant differences in terms of temperature threshold and average conduction velocities. An understanding of the temperature response properties of afferent neurons innervating the paw skin have been critical in many mechanistic discoveries, some leading to new pain therapies. A clear understanding of trigeminal nociceptors may be similarly useful in the investigation of trigeminal pain mechanisms and potential therapies.

Identifying biological markers of activity in human nociceptive pathways to facilitate analgesic drug development

Nearly 20% of the adult population in Europe suffers from moderate to severe chronic pain and only half receive beneficial treatment [8]. In US, 37% of younger adults and 57% of older adults suffer with pain lasting at least a year [9]. Despite the significance of this problem and advances in our understanding of pain mechanisms, the pace at which new treatments become available to chronic pain patients has been very slow. In recent decades, successful novel analgesics have been mostly found through serendipity or refinement of existing approaches (e.g., gabapentin, cyclooxygenase-2 and monoamine reuptake inhibitors). Analysis of drug development failures indicates that they occur primarily in clinical phases, and are mostly due to a lack of translation of efficacy in animal models to patients [7,22]. Although a comprehensive analysis of problems in analgesic development is beyond the scope of this review, we hypothesise that the preclinical to clinical transition can be facilitated by a rational use, in early drug development stages, of biological markers of activity in human nociceptive pathways. We also propose that such markers could have translational value if adapted for animal studies to provide efficacy measures more clinically relevant than withdrawal reflexes, see [29].

Non-invasive diode laser activation of transient receptor potential proteins in nociceptors.

We investigated diode laser (980 nm) evoked activation of transient receptor potential proteins (TRPV1 and TRPV2). C and A-delta (A&dgr;) nociceptor families are primarily responsible for pain mediation in the peripheral nervous system. TRPV1 proteins have been associated with heat evoked pain in C fibers while A&dgr; fibers have been associated with TRPV2. Diode laser stimulation allows a margin of safety between non-invasive activation and damage 19, 22, 34. Laser pulses (20-50 ms, 0.1-10 W, 980 nm) were used to stimulate: A) in vitro: excised patches from HEK293 cells expressing TRPV1; B) in vitro: rat DRG nociceptors expressing either TRPV1 or TRPV2; and C) in vivo: C-fibers of the rat saphenous nerve (SN) trunk. Cell currents were recorded using standard patch clamp methods. The SN was also stimulated electrically with bipolar electrodes. Stimulation (20-50 ms) of HEK and DRG cells expressing TRPV1 was highly reproducible. Activation and peak currents were achieved at estimated peak temperatures of 55°C and 70°C. Threshold activation was also observed in DRG neurons expressing TRPV2. The conduction velocity for laser-activated saphenous nerve afferents was in the C fiber range (0.5-1 m/s). Electrically stimulated nerve contained stimulation artifacts and complex neural components with conduction velocities ranging from 0.3-30 m/s. Diode laser activation of TRPV1 protein is a reproducible and effective means to probe TRP activity in both in vivo and in vitro preparations

Sensory Small Fiber Function Differentially Assessed with Diode Laser (DL) Quantitative Sensory Testing (QST) in Painful Neuropathy (PN)

Sensory function of small peripheral nerve fiber was assessed by means of quantitative sensory testing (QST) during which sensory stimulation was provided using diode laser (DL) in patients suffering from painful neuropathy (PN) and compared with symptom-free healthy controls (HC). Based on previous research work using DL stimulation, parameters that demonstrated safe and specific activation of A-delta, which were distinct from stimulation parameters for the activation of C-fibers, were utilized in this study. Results of this study demonstrated that this differential activation pointed to the impaired function of A-delta fibers while C-fiber function was unaffected. Stimulation of HC reproduced previously published results, and stimulation during this study was safe also without any dermal effect in patients with PN and in HC. Parameters used in this study were demonstrated in previous preclinical rodent study identical differential effect on activation of A-delta and C-fibers, and as such, DL is an ideal tool for translational pain research where specific activation of A-delta or C-fibers, or both, is required.