Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia

Abstract

Resiniferatoxin (RTX) is an ultrapotent capsaicin analog that binds to the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). There is a large body of evidence supporting a role for TRPV1 in noxious-mediated and inflammatory hyperalgesic responses. In this study, we evaluated low, graded, doses of perineural RTX as a method for regional pain control. We hypothesized that this approach can provide long-term, but reversible, blockade of a portion of nociceptive afferent fibers within peripheral nerves when given at a site remote from the neuronal perikarya in the dorsal root ganglia. Following perineural RTX application to the sciatic nerve, we demonstrated a significant inhibition of inflammatory nociception that was dose- and time-dependent. At the same time, treated animals maintained normal proprioceptive sensations and motor control, and other nociceptive responses were largely unaffected. Using a range of mechanical and thermal algesic tests, we found that the most sensitive measure following perineural RTX administration was inhibition of inflammatory hyperalgesia. Recovery studies showed that physiologic sensory function could return as early as two weeks post-RTX treatment, however, immunohistochemical examination of the DRG revealed a partial, but significant reduction in the number of the TRPV1-positive neurons. We propose that this method could represent a beneficial treatment for a range of chronic pain problems, including neuropathic and inflammatory pain not responding to other therapies.

Figures

Figure 1

Locating the sciatic nerve for electrically-stimulated percutaneous, perineural RTX injection. Note finger palpation locations and direction of the needle (see Methods). Fluorescein dye injection (50 μl, 1 μg/μl) using this technique demonstrates the distribution of dye-containing fluid within the perineurium of the nerve (B).

Figure 2

Percutaneous application of RTX to the sciatic nerve and sciatic plus saphenous nerves minimally effects response to heat stimuli. RTX (250 ng, 50 μl) application to only the sciatic nerve produced a transient change in heat withdrawal latency with respect to testing time (*P < 0.05, 1 way ANOVA, Scheffe post-hoc analysis), as compared to the baseline. Additional application of RTX to the saphenous nerve produced a modest increase in heat latency, but this effect was not significantly different, as compared to the sciatic group. The "sciatic nerve" group refers to animals treated only with percutaneous RTX (N = 24) and the "sciatic and saphenous nerves" group refers to animals that were injected percutaneously near the sciatic nerve and had an open field injection around the saphenous nerve (N = 15). The "control group" represents the uninjected right hindpaw. There was no difference between contralateral controls for the sciatic nerve group (N = 24) and the sciatic/saphenous group (N = 15), therefore these groups were combined (N = 39) for subsequent comparisons.

Figure 3

RTX blocks inflammatory heat hyperalgesia when applied directly to the sciatic nerve. Both percutaneous and open field injection of RTX (250 ng, 50 μl) inhibited inflammatory heat hyperalgesia following carrageenan injection (6 mg, 150 μl). There was no significant difference in latency time regarding RTX application method (i.e. percutaneously vs. direct open field application); therefore data were pooled (N = 15). However, for this set of animals, there was a significant increase in normal heat latency as compared to vehicle treated animals (*P < 0.05, 1 way ANOVA, Scheffe post-hoc analysis). Animals pretreated with vehicle (0.25% Tween-80, PBS 50 μl, N = 10) had a significant decrease in their heat withdrawal latency (+P < 0.05, 1 way RM-ANOVA) following induction of inflammation.

Figure 4

Percutaneous RTX produces long-lasting, reversible inflammatory heat hyperalgesic inhibition (A) in a dose-dependent fashion (B). Inflammatory heat hyperalgesia is significantly inhibited 1 day (N = 8), 1 week (N = 6), and 2 weeks (N = 5) following application of RTX (250 ng, 50 μl) to the sciatic nerve (*P < 0.05, 1 way ANOVA). An intermediate response was seen at 1 month (N = 8), and at 3 months (N = 8). Complete recovery with a normal heat withdrawal response following inflammation was observed 6 months (N = 11) following treatment, indicating that the effect of RTX had reversed. The dose-response results (B) indicate that ≥ 125 ng is necessary for a significant anti-inflammatory effect, as compared to Pre-Inflammation values and to vehicle (P < 0.05). Note that the Pre-inflammation testing point represents the same testing day that the animal was to be inflamed. There was no difference between groups treated with RTX (N = 46); therefore data were pooled for the baseline (Baseline) and Pre-inflammation testing sessions. Animals treated with vehicle (N = 16) were also pooled in the Baseline, Pre-inflammation, and Post-inflammation groups for comparison.

Figure 5

RTX does not affect normal mechanical sensitivity or rotarod performance. Withdrawal force latencies were similar for vehicle and RTX treated animals except after inflammation, where there was a significant difference (*P < 0.05, Mann-Whitney rank sum test) between the two groups (A). Data presented as a median normalized threshold (bars: interquartile range). Pre-inflammation values for both vehicle and RTX-treated groups were all significant versus the post-inflammation value (Krukal-Wallis ANOVA, P < 0.05). Values are represented by a median normalized threshold and the interquartile range is denoted by the bars. Rotarod performance was identical for both groups (B).

Figure 6

Dorsal root gangliaTRPV1 immunopositive cells are eliminated following perineural RTX treatment. Lumbar dorsal root ganglia sections (7 μm, paraffin-embedded) were stained for TRPV1 following RTX, vehicle or no treatment of the sciatic nerve. There was a significant difference in the number of TRPV1 positively stained cells in the RTX-treated animals, as compared to vehicle and untreated animals (Table inset).

Figure 7

RTX blocks capsaicin-induced efferent neurogenic inflammation. Following Evans blue injection (i.v., 30 mg/kg, 2% solution), a 1% capsaicin cream was liberally applied to the shaved hind legs and paws. Areas of neurogenic-induced plasma extravasation are seen as blue. Note the delineation between the blue area (positive for plasma extravasation) versus the white skin (negative for plasma extravasation) for the RTX treated side (A, C) and extravasation on only the three medial toes for the RTX-treated side. Perineural vehicle application did not effect plasma extravasation (B). These data indicate selective targeting of the sciatic nerve while preserving the saphenous division via this percutaneous injection.