Resiniferatoxin (RTX) causes a uniquely protracted musculoskeletal hyperalgesia in mice by activation of TRPV1 receptors

Abstract

Inactivation of transient receptor potential vanilloid-1 (TRPV1) receptors is one approach to analgesic drug development. However, TRPV1 receptors exert different effects on each modality of pain. Because muscle pain is clinically important, we compared the effect of TRPV1 ligands on musculoskeletal nociception to that on thermal and tactile nociception. Injected parenterally, capsaicin had no effect on von Frey fiber responses (tactile) but induced a transient hypothermia and hyperalgesia in both the tail flick (thermal) and grip force (musculoskeletal) assays, presumably by its agonistic action at TRPV1 sites. In contrast, resiniferatoxin (RTX) produced a chronic (>58 days) thermal antinociception, consistent with its reported ability to desensitize TRPV1 sites. In the same mice, RTX produced a transient hypothermia (7 hours) and a protracted (28-day) musculoskeletal hyperalgesia in spite of a 35.5% reduction in TRPV1 receptor immunoreactivity in muscle afferents. Once musculoskeletal hyperalgesia subsided, mice were tolerant to the hyperalgesic effects of either capsaicin or RTX whereas tolerance to hypothermia did not develop until after 3 injections. Musculoskeletal hyperalgesia was prevented but not reversed by SB-366791, a TRPV1 antagonist, indicating that TRPV1 receptors initiate but do not maintain hyperalgesia. Injected intrathecally, RTX produced only a brief musculoskeletal hyperalgesia (2 days), after which mice were tolerant to this effect.

Perspective: The effect of TRPV1 receptors varies depending on modality and tissue type, such that RTX causes thermal antinociception, musculoskeletal hyperalgesia, and no effect on tactile nociception in healthy mice. Spinal TRPV1 receptors are a potential target for pain relief as they induce only a short musculoskeletal hyperalgesia followed by desensitization.

Keywords: Capsaicin; desensitization; musculoskeletal hyperalgesia; nociception; pain.

Figure 1

A single injection of capsaicin or RTX decreases grip force but causes variable responses in tail flick latencies, von Frey fiber responses and body temperature. Mice were injected s.c. with 2 mg/kg of capsaicin (A, D, G, J), 0.02 mg/kg of RTX (B, E, H, K), or 0.1 mg/kg of RTX (C, F, I, L). Grip force (A, B, C), tail flick (D, E, F), von Frey (G, H, I) measurements, and body temperature (J, K, L) were recorded. Statistical analyses were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis where the asterisk indicates P<0.05 when compared to vehicle.

Figure 2

Unlike capsaicin, multiple injections of RTX cause tolerance to its effect on grip force but variable effects on tail flick and body temperature. Mice were injected daily with 2 mg/kg of capsaicin (A, D, F), a low dose (0.02 mg/kg) of RTX every 6–10 days (B, E, G), or high dose (0.1 mg/kg) of RTX (C, H). Grip force (A, B, C) and tail flick (D, E) and body temperature (F, G, H) measurements were recorded after each injection, as indicated. Statistical analyses were performed using the unpaired Student’s t-test where the asterisk indicates P<0.05 when compared to vehicle on the same day.

Figure 3

Repeated injections of a low dose of RTX decreases sensitivity of TRPV1 receptors to capsaicin and to RTX. Animals were injected s.c. 4 times with 0.02 mg/kg of RTX at 6- to 10-day intervals and 5 days later challenged with either 2 mg/kg of capsaicin (A, C) or 0.1 mg/kg of RTX (B, D). Grip force (A,B) and tail flick (C, D) measurements were taken 30 min after capsaicin or 24 h after RTX. Statistical analyses were performed using a two-way ANOVA followed by Bonferroni’s post hoc analysis where the asterisk reflects P<0.05 when compared to the values indicated.

Figure 4

Morphine prevents and reverses the decrease in grip force produced by capsaicin (A) or RTX (B). Grip force measurements were taken 45 min after the i.p injection of morphine and 30 min after the s.c. injection of either capsaicin (2 mg/kg) or 24 h after the injection of RTX (0.1 mg/kg). Statistical analyses were performed using a one-way ANOVA followed by Newman-Keuls’ post hoc analysis where the asterisk represents P<0.05, when compared to the values indicated.

Figure 5

TRPV1 antagonist (SB-366791) prevents the musculoskeletal hyperalgesia produced by capsaicin and by RTX. Animals were injected with SB-366791 (0.5 mg/kg i.p.) and 20 min later they were injected s.c. with either 2 mg/kg of capsaicin (A) or 0.1 mg/kg of RTX (B). Grip force was measured 30 min after capsaicin and 24 hr after RTX. In panel C, mice were injected with SB-366791 at this same dose 8 days after the injection of RTX and grip force was measured at the times indicated. Statistical analyses were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis where the asterisk indicates P<0.05 when compared to all the other values at that time.

Figure 6

Intrathecal (i.t.) injection of RTX produced a transient (2 day) musculoskeletal hyperalgesia. Mice were injected i.t. with 0.125 μg/animal of RTX or vehicle and grip force (A), von Frey fiber (B) and tail flick (C) responses were measured at the times indicated. Statistical analyses were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis where the asterisk indicates P<0.05 when compared to vehicle controls measured at the same time.

Figure 7

Intrathecal (i.t.) injection of RTX resulted in tolerance to the hyperalgesic effect of RTX injected i.t. or s.c. In panel A, mice were injected i.t. with 0.125 μg/animal of RTX or vehicle 4 days prior to a second challenge with the same i.t. dose of RTX or vehicle and grip force measured 24 h after each injection. In panel B, mice were injected with 0.125 μg/animal of RTX i.t. and 4 days later challenged with 0.1 mg/kg of RTX or vehicle injected s.c. and grip force measured 24 h later. Statistical analyses were performed using an unpaired Student’s t-test where the asterisk indicates P<0.05 when compared to vehicle-injected controls.

Figure 8

RTX decreased TRPV1 receptor-immunoreactivity in afferents projecting to skeletal muscle without causing their degeneration. Mice were injected with fluorogold (FG 4%, 10 μL in the gastrocnemius muscle) either 5 days before (FG→RTX) or 2 days after (RTX→FG) injection of RTX (0.1 mg/kg s.c.). Representative pictures from an L4 DRG of a vehicle-injected mouse showing cells that are (A) retrogradely labeled with fluorogold (green), (B) immunoreactive for TRPV1 receptors (red), (C) and double-labeled for both fluorogold and TRPV1 receptors (Yellow). Arrows indicate fluorogold-labeled cells that are also immunopositive for TRPV1 receptors. Scale bar=20 μ. The total number of FG-labeled projections in the DRG (L3–L5) was counted (D), as well as the percentage of FG projections that were TRPV1 receptor-immunopositive (E). Statistical analyses were performed using a one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc analysis where the asterisk indicates P<0.05 when compared to vehicle.