Ablation of Rat TRPV1-Expressing Adelta/C-Fibers with Resiniferatoxin: Analysis of Withdrawal Behaviors, Recovery of Function and Molecular Correlates

Abstract

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.

Transforming Growth Factor-β1 Regulates Cdk5 Activity in Primary Sensory Neurons

“…Cutaneous afferents were activated either by low-rate (Յ1.5°C) heating using long pulses, low energy, and a large diameter beam (5 mm Ø, nominal) or by a high rate (Ն150°C) of heating, using a high-energy, short pulse (100 ms), and a small beam diameter (1.6 mm Ø, nominal). Under these parameters, long pulses preferentially activate C-fibers, and short pulses preferentially activate A␦ fibers (32,33). Longpulse responses were evaluated at three laser current settings, 650, 750, and 850 mA, and short-pulse responses were evaluated at 2500, 3000, 3500, and 4000 mA.…”

“…We could not detect any change in both p35 and Cdk5 protein levels in DRG and TG, as well as in Cdk5 activity in TG and DRG from postnatal 45-day-old Tgfbr1 cKO compare with control mice (data not shown). To evaluate whether pain sensation was affected in Tgfbr1 cKO mice, acute nociceptive responses were measured using an infrared diode laser to activate cutaneous thermal nociceptive nerve endings (33) in the hind paws of Tgfbr1 cKO mice between 1 and 2 months of age, who showed no signs of inflammation or body FIGURE 3. TGF-␤1 increased TRPV1 phosphorylation resulting in increased intracellular Ca 2؉ influx in DRG primary culture.…”

Positive allosteric modulation of TRPV1 as a novel analgesic mechanism

“…Administration of a vanilloid agonist, such as capsaicin or its ultrapotent analog RTX [31], can cause calcium-induced cytotoxicity and lead to a TRPV1-selective axonopathy that spares surrounding non-TRPV1-expressing somatosensory proprioceptive afferent and motor efferent nerve fibers. The potency and selectivity of vanilloid agonists for TRPV1 afferents has been demonstrated repeatedly in vivo [8,[32][33][34][35][36][37][38][39][40]. For example, ablation of TRPV1 + nerve terminals occurring after a single subcutaneous injection of RTX into the rat hind paw results in prolonged but reversible analgesia that can be detected for one to several weeks and induces up-regulation of molecular markers for axon damage/repair in neuronal perikarya of the dorsal root ganglia [8,32].…”

“…The potency and selectivity of vanilloid agonists for TRPV1 afferents has been demonstrated repeatedly in vivo [8,[32][33][34][35][36][37][38][39][40]. For example, ablation of TRPV1 + nerve terminals occurring after a single subcutaneous injection of RTX into the rat hind paw results in prolonged but reversible analgesia that can be detected for one to several weeks and induces up-regulation of molecular markers for axon damage/repair in neuronal perikarya of the dorsal root ganglia [8,32]. We have also found that topical application of RTX onto the cornea results in temporary loss of the capsaicin eyewipe response in parallel with a loss of CGRP immunoreactive afferents in stromal fiber bundles [41].…”

“…Given the detrimental side effects and addiction potential of these medications, it is critical that we develop novel, nonopiate based pharmacological agents to treat chronic pain. One potential therapeutic avenue involves targeting TRPV1, a sodium/calcium ion channel highly expressed in a subpopulation of unmyelinated C-and lightlymyelinated Aδ-afferent primary afferent neurons [6][7][8][9]. This receptor responds to multifactorial inputs and is activated by temperature >43°C [7], protons [7], exogenous vanilloid ligands such as capsaicin and resiniferatoxin (RTX) [7], endogenous vanilloids such as NADA [10], the endocannibinoid anandamide [11,12], and various fatty acids [13][14][15].…”