Ation of IM is actually a well-established preclinical model of headache [372]. First, we modified

Ation of IM is actually a well-established preclinical model of headache [372]. First, we modified the composition of IM and applied it onto the dura of well-habituated adult male mice. The home-cage behavior of mice receiving Fluorometholone In Vitro automobile or IM was observed for 2 h. Dural Yohimbic acid References application of IM elicited robust forepaw wiping and hindpaw scratching around the scalp and periorbital area within the V1 dermatome. The duration of wiping and scratching peaked 400 min immediately after IM exposure and progressively subsided (Figure 7a). Mice that received dural IM application exhibited drastically longer duration of wiping and scratching than mice treated with automobile (Figure 7b, p 0.001, two-tailed t-test), suggesting that meningeal irritation elicits ongoing nocifensive behavior in adult mice. Subsequent, we co-applied two.eight mM TRPM8 agonist (-)-menthol as well as the vehicle or IM onto the dura andaPb9 eight 7 six five four three two 1Axon Density (mm-1)Cornea Dura###25change of axon densityAdultcPAdult80 60 40 20 0 -20 -40 -CorneaEGFPf+DuraFigure 6 Postnatal boost within the EGFPpositive fiber density in the corneal epithelium of TRPM8 mice. a Representative photos of axons containing EGFPir inside the basal epithelium of cornea in P2 and adult TRPM8EGFPf+ mice. b EGFPpositive fiber densities in the corneal epithelium of P2 and adult TRPM8EGFPf+ mice (n = 7 and five mice, respectively). The EGFPpositive fiber densities inside the dura of P2 and adult TRPM8EGFPf+ mice are also plotted (same data as in 5a). p 0.01, p 0.001, twoway ANOVA with post hoc Bonferroni test. ###p 0.001, compared with all the P2 dura group. c Percentage transform of EGFPpositive axon density from P2 to adulthood inside the cornea and dura of TRPM8EGFPf+ mice (identical mice as in b). The percentage transform is calculated as (adultdensity – P2density)P2density 100. p 0.001, twotailed ttest.Ren et al. Mol Pain (2015) 11:Web page 9 ofaDuration of wiping and scratching (sec)Duration of behavior (sec)140 120 one hundred 80 60 40 20 0 0 20 40 60 80vehicle IM naiveb500 400 300 200 100Time (min)vehicleIMcDuration of behavior (sec)600 500 400 300 200 one hundred menthol AMTB-+–+-+-+ +vehicleIMFigure 7 Dural application of TRPM8 agonist ()menthol inhibits meningeal irritationinduced ongoing nocifensive behavior in adult mice. a Time spent on forepaw wiping and hindpaw scratching about the scalp and periorbital location (within trigeminal V1 dermatome) in 20 min bins in response to dural application of automobile or IM in adult male mice (n = 12 and 9, respectively). Na e mice (n = six) have been habituated to the test room and recording cage as mice in other groups but had been not subjected to anesthesia exposure, surgery or drug application. b Total duration of nocifensive behavior through the 120 min recording period in mice that received dural application of car or IM (exact same mice as in a, p 0.001, twotailed ttest). c Dural application of ()menthol (2.eight mM in 20 ) reduces the duration of vehicle and IMinduced nocifensive behavior (n = 6 mice in each group; p 0.001, twoway ANOVA general impact, p 0.01, p 0.001, post hoc Bonferroni test amongst individual groups). Co application of menthol and TRPM8 antagonist AMTB (2.eight mM in 20 ) reverses the effect of menthol (n = 3 mice; p 0.01, p 0.001). AMTB doesn’t alter the duration of IMinduced nocifensive behavior (p = 0.72, among IM and IM+ AMTB groups, n = 6 and 3 mice, respectively).recorded the duration of nocifensive behavior. Previous studies show that topical application of 1 mM (-)-menthol produces analgesic effects exclusively.