Nociception, neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice

Abstract

Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice. Thermoregulatory behaviour in relation to environmental thermopreference (30 vs. 35°C) was slightly impaired in male knockdown mice, but the reduction of TRPV1 function was not associated with enhanced hyperthermia. TRPV1 agonist resiniferatoxin induced hypothermia and tail vasodilatation was markedly inhibited in knockdown mice. In conclusion, shRNA-mediated knock down of the TRPV1 receptor in mice induced robust inhibition of the responses to TRPV1 agonists without altering the expression, gating function or neurogenic oedema provoked by TRPA1 activation. Thermoregulatory behaviour in response to heat was inhibited, but enhanced hyperthermia was not observed.

Fig. 1

Chemonociception induced by instillation of capsaicin (10 μg/ml in saline) into the eye of mice carrying the shRNAa transgene. The numbers of protective eye wiping movements of tg+ progeny of founder animals nos. 4 and 11 are shown. The bars represent mean ± SD values of five experiments. ***P < 0.001 compared with wild type (FVB/Nhsd) mice, one-way ANOVA test, Dunnett’s post hoc comparison

Fig. 2

a Measurement of TRPV1 and TRPA1 expression in TRG neurons of tg− and tg+ mice by TaqMan quantitative PCR assay. ΔC _t values normalized to housekeeping genes are shown. Bars represent mean ± SD values of three experiments. ***P < 0.001, two-way ANOVA test, Bonferroni post hoc comparison. b Capsaicin and mustard-oil sensitivity of trigeminal sensory neurons isolated from tg− and tg+ mice, measured by fura-2 microfluorimetry. The percentages of cells responsive (ΔR _F340/F380 > 0.1) to capsaicin or mustard oil treatment are shown. ***P < 0.001, Fisher’s exact test

Fig. 3

Capsaicin-induced nocifensive behaviour on the paw of tg− and tg+ mice. Capsaicin (20 μl, 100 μg/ml) was injected into the sole of the left hindpaw and licking and flinches were observed and a composite pain score was calculated. Results are shown as means ± SD of the sum of pain score in seconds (n = 8–16 animals per group). In tg+ mice there was no significant difference between responses evoked by capsaicin or by the solvent (P > 0.05). ***P < 0.001, two-way ANOVA test, Bonferroni post hoc comparison

Fig. 4

Neurogenic ear oedema in tg− and tg+ mice. a Topical application of 2.5% capsaicin. The solvent of capsaicin (96% ethanol) was used as control. The results are expressed as percent swelling as compared to the initial (0-h) control. The data points represent the mean ± SD values of five to seven experiments. ***P < 0.001, capsaicin treatment, tg+ versus tg− animals, two-way ANOVA test, Bonferroni post hoc comparison. b Topical application of 1% mustard oil. The solvent of mustard oil (paraffin oil) was used as control. Results are expressed as percent swelling as compared to the initial (0-h) control. The data points represent the mean ± SD values of five to seven experiments. There was no significant difference between tg+ and tg− animals (P > 0.05, two-way ANOVA test, Bonferroni post hoc comparison)

Fig. 5

a Temperature preference (30 or 35°C) of male and female tg− and tg+ mice was measured at 10-min intervals. The percentage of time spent at 35°C is shown. The data points represent the mean ± SD of eight mice in each group. *P < 0.05, male tg+ versus tg−, two-way ANOVA test, Bonferroni post hoc comparison. b Rectal temperatures of the animals measured at the beginning and at the end of the temperature preference experiment. The data points represent the mean ± SD of eight experiments. The only significant difference was observed in male mice at the end of the experiment (*P < 0.05, two-way ANOVA test, Bonferroni post hoc comparison)

Fig. 6

a RTX-induced hypothermia in tg− and tg+ mice. Body temperature was measured before i.p. injection of 20 μg/kg RTX and at 7.5, 15, 30 and 60 min after injection. The solvent of RTX was used as control. The data points represent the mean ± SD of ten mice in each group. *P < 0.05, ***P < 0.001, RTX-treated tg+ versus tg− mice, two-way ANOVA test, Bonferroni post hoc test. b RTX-induced change in blood flow in the tail of tg−, tg+ and TRPV1 KO mice. After induction of anaesthesia with urethane, mice received an i.p. injection of 20 μg/kg RTX and blood flow in the tail was monitored by laser doppler imaging every third minute. The percentage change in blood flow is shown compared to the initial values measured at the beginning of the experiment. *P < 0.05, **P < 0.01, tg+ versus tg− animals, two-way ANOVA test, Bonferroni post hoc test