Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors

Abstract

The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26 °C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor.

Fig. 1

Effect of a cold swim stress on musculoskeletal nociception, body temperature and tail flick latency. Swim stress (15-min at 26°C) decreased grip force (A) simultaneously with a decrease in core body temperature (C) and an increase in tail flick latency (E) when measured before and at 0, 15 and 45 min after the end of the swim. Panels on the right depict the effect of a cold swim of different durations (5, 15 and 30 min) on grip force responses (B), body temperature (D) and tail flick latencies (F). Throughout the figures, SEM is calculated for all points but not shown by our graphical representation when smaller than the width of the symbol. Statistical analyses of each panel were performed using a two-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis. The asterisk indicates P<0.05 when the swim group was compared to the no swim group at a specific time before or after the swim (A,C,E). The effect immediately after swims of 3 different durations were compared to each group’s values before the swim using a paired Student’s t-test and then control values were pooled for graphical representation (B,D,F). The number sign further indicates P<0.05 when a group was different from all other values in that panel when compared using ANOVA, as described above. Throughout the figures, the number of animals used in each group is indicated in the key or at the base of each bar.

Fig. 2

Effect of morphine and of RTX on swim stress-induced decreases in grip force values. Grip force was measured before and after a 15-min swim in 26°C water. Morphine was delivered at a dose of 10 mg/kg i.p. 15 min prior to the onset of the swim (A) or before the tail flick assay (51°C) (B). Also in the lower panel, RTX was delivered at a dose of 0.125 µg i.t. 24 hr before testing in the tail flick assay (B). Statistical analyses on panel A were performed using a two-way ANOVA followed by Bonferroni’s post hoc analysis. Significant differences (P<0.05) between groups are indicated by an asterisk. In panel B, statistical analyses were performed using unpaired Student’s t-test where the asterisk indicates P<0.05 when compared to vehicle.

Fig. 3

Effect of antagonism of CRF1 and CRF2 sites on the cold stress-induced decrease in grip force. Panel A illustrates the effect of NBI-35965 (NBI), a CRF1 antagonist, on grip force, while panel B illustrates the effect of astressin 2B, a CRF2 receptor antagonist. Each drug was injected 15 min prior to the onset of a 15-min cold (26°C) swim. Panel C shows that the chosen dose of NBI-35965 (50 µg/5 µl i.c.v.) was physiologically active, as it was sufficient when injected 30 min prior to a swim stress to prevent the increase in plasma corticosterone, a CRF1 receptor-mediated effect. Similarly, in a separate experiment, 20 µg/5 µl of astressin 2B was injected i.t. and found to prevent the decrease in body temperature at 4 h after injection of 10 µg/5µl of urocortin I, a CRF2 receptor-mediated effect (D). Statistical analyses were performed in panels A and B using a two-way ANOVA followed by a Bonferroni post hoc analysis. Data in panels C and D were analyzed using an unpaired Students t-test (swim to no swim), and a one-way ANOVA followed by Newman-Keuls post hoc analysis comparing data after the swim in panel C and all data in panel D. Significance (P<0.05) indicated by an asterisk.

Fig. 4

Effect of desensitization and antagonism of TRPV1 receptors on grip force responses measured before and immediately after each swim. Mice were subjected to a 15-min swim in cold (26°C) (A) or in warm (41°C) water (C) after pretreatment with a desensitizing dose of RTX injected 6 days previously (0.125 µg) or 17 days previously (0.1 mg/kg s.c). Separate groups of mice were swum in cold (B) or warm water (D) 15 min after pretreatment with SB-366791 given either i.t. (30 µg/mouse) or i.c.v (30 µg/mouse), or 30 min after SB-366791 delivered i.p. (0.5 mg/kg). Statistical analyses were performed using a paired Student’s t-test comparing the effect of each drug to its vehicle-injected control group. The lower two panels (E,F) demonstrate the effect of the doses of RTX and of SB-366791 used in panels A and C in additional groups of mice. Compared to vehicle-injected control mice, a single injection of RTX (0.125 µg/mouse i.t.) decreased grip force values 24 hr later, but 4 days later the hyperalgesic effect of a second injection of this dose of RTX was absent in RTX-pretreated mice, but not in those pretreated with vehicle. This illustrates the development of desensitization to the hyperalgesic effect of RTX (E). SB-366791 (0.5 mg/kg i.p.) 20 min before an injection of capsaicin (2 mg/kg s.c.) prevented capsaicin-induced decreases in grip force values (F). Statistical analyses were performed using a one-way ANOVA followed by Newman-Keuls post hoc analysis (E,F). Significant differences (P<0.05) are shown by the asterisk, as indicated. The number sign indicates P<0.05 when a group was different from all others.

Fig. 5

Effect of 15 daily swims on grip force responses, body temperature and tail flick latencies. The magnitude of the grip force responses taken immediately after a daily 15-min swim (26°C) were compared to values from groups not subjected to the daily swim (A). Each day, grip force responses returned to their pre-injection control values by 1 hr or less after the termination of each swim. Body temperature (B) and tail flick latencies (51°C) were also measured immediately after the 15-min swim (C). Statistical analyses of each panel were performed using a two-way ANOVA followed by Bonferroni post hoc analysis where the asterisk indicates P<0.05 when compared to vehicle-injected controls.