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

Abstract

Background: Ablation 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.

Results: RTX 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.

Conclusion: Behavioral 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.

Figures

Figure 1

Detection of early-onset nocifensive behaviors prior to withdrawal of stimulated limb. A 100 msec thermal pulse at high power (6.08 W/mm2) was used to stimulate the right index toe. Withdrawal responses were recorded at a rate of 500 frames per second. The position of the rat's head before and after stimulation are highlighted in A and B, respectively. The upper and lower arrowheads in each frame bring attention to the eyes and nose, respectively. Abrupt head displacement in (A) versus (B) occurs by 110 msec (140-30 msec, see Methods). Also note in B the orbital "tightening", which was observed only with some rats following laser stimulation. (C) By 210 msec, the head was oriented towards the stimulated foot and toes on the stimulated foot have moved. (D) Withdrawal of the stimulated paw was underway by 310 msec. (E) and (F) are magnifications of (A) and (B), respectively, showing that by 110 msec, toe movement on the non-stimulated hind paw has preceded any movement detected in the stimulated limb. Full videos can be viewed in the Additional file 1 Movie S1 data and show that latency to the first detectable movement was approximately 74 msec.

Figure 2

Effect of RTX on Aδ and C responses at the mid-plantar injection site. Either 5 or 50 ng of RTX was injected into the right while vehicle was injected into the left hind paw. Aδ and C responses before and after injections were followed for several weeks. (A-D and F) Aδ responses following intraplantar RTX-injection. We used probability of withdrawal (A, C and F) and the intensity of withdrawal (B and D) as endpoints for the Aδ assay. Behavioral responses to stimulus intensities at 5.12 W/mm2 (A, B and F) or 6.08 W/mm2 (C and D) are shown. (E) Loss and return of C-fiber responses after RTX-intraplantar injection. We used latency to paw withdrawal as the endpoint for the C-fiber assay; a stimulus intensity was chosen that produced a response latency of ~8 sec in normal rats. A cutoff of 16 sec (dashed line) was imposed to prevent tissue damage. (F and G) Direct comparison of behavioral responses at day 18 post-RTX to Aδ (F) and C (G) stimuli. Data used in week 3, in the line graphs, are the average of tests done on days 16, 18 and 21. Hypoalgesia at day 18 was maintained in Aδ but not C-fibers. The tables under the graphs denote statistical comparisons between vehicle and 5 ng or 50 ng RTX, which are aligned with the post-treatment time points. Each data point is an average of the combined responses from 2 or 3 non-consecutive days of testing during the time period indicated on the x-axis (weeks). For graphical simplicity, the data from all vehicle-treated rats (n = 12) is combined, yielding a single line in the plots, since there was no significant difference between the two vehicle groups. 2-way ANOVA with repeated measures was used to compare the effect of 5 ng or 50 ng RTX versus vehicle. 2-way ANOVA was used to compare the effects of 5 ng versus 50 ng. Fisher's exact test was used in F. *,† p < 0.5, **,†† p < 0.01, ***, ††† p < 0.001, n = 6 rats per group.

Figure 3

Aδ- and C-fiber responses in the toes after mid-plantar RTX injection. The infrared diode laser was used to stimulate Aδ- or C-fibers in the toes of the same rats used in Figure 2. (A-D) Responses to Aδ stimuli are diminished for up to 10 weeks. Plots are similar to those in Figure 2. (E) For 50 ng RTX only, a small but significant difference was measured in the toes at 5 weeks (but not at 10 weeks) for C responses; data reported are an average of 3 test days. Our initial protocol used C-fiber testing only at the mid-plantar footpad; however, since we observed prolonged, diminished Aδ behavioral responses in toes after RTX-treatment, a suppression of C-fiber behavioral responses might occur in tandem. During a separate study, we found that within 1 week after 50 ng intraplantar RTX, rats reached the 16 sec cutoff when testing C-fibers in their toes. 2-way ANOVA with repeated measures was used to compare the effect of 5 ng or 50 ng RTX versus vehicle. 2-way ANOVA was used to compare the effects of 5 ng versus 50 ng. *,† p < 0.5, **,†† p < 0.01, ***,††† p < 0.001, n = 6 rats per group.

Figure 4

Intraplantar injection of RTX leads to a transient increase in gene markers of nociception but a sustained increase in markers of nerve regeneration. Gel image shows ganglionic expression levels of mRNA encoding MCP-1 and ATF3 24 h or 10 days after vehicle or RTX treatment, taken from left and right L4-L5 dorsal root ganglia (n = 4 rats, ipsilateral RTX expression is denoted by an asterisk). MCP-1 (C) and ATF3 (D) transcript levels were normalized to GPDH. Data were obtained from RT-PCR analysis (n = 4/group). All graphs are presented as mean ± SEM. *p < 0.05 and **p < 0.01, and ***p < 0.001 as determined by a one-way ANOVA followed by a Bonferroni correction. (E-H) Immunohistochemistry showing spinal cord c-Fos expression 6 h or 10 d post-RTX. E and F are epifluorescent images depicting an elevation in c-Fos protein 6 h after RTX or vehicle in the ipsilateral and contralateral dorsal horn, respectively. G and H are epifluorescent images representing c-Fos expression 10 d after RTX or vehicle in the ipsilateral and contralateral dorsal horn, respectively. (I) Cell counts demonstrate significantly more c-Fos immunopositive cells in spinal cord ipsilateral to RTX injection as compared to contralateral vehicle injections at 6 h but not at 24 h or 10 days. Data expressed as means ± SEM (n = 4). The fold-change is shown. *p < 0.05 as determined by Student's t-test. Arrows indicate the location of the medial dorsal horn. The most intense c-Fos positive neurons were counted in this area.