Ation of IM can be a well-established preclinical model of headache [372]. Very 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 vehicle or IM was observed for two h. Dural application of IM elicited robust forepaw wiping and hindpaw scratching around the scalp and periorbital area within the V1 dermatome. The Benzylideneacetone Phospholipase duration of wiping and scratching peaked 400 min 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 2.8 mM TRPM8 agonist (-)-menthol in addition to the automobile or IM onto the dura andaPb9 8 7 six five four three two 1Axon Density (mm-1)Cornea Dura###25change of axon densityAdultcPAdult80 60 40 20 0 -20 -40 -CorneaEGFPf+DuraFigure six Postnatal improve within the EGFPpositive fiber density within the corneal epithelium of TRPM8 mice. a Representative images of axons containing EGFPir inside the basal epithelium of cornea in P2 and adult TRPM8EGFPf+ mice. b EGFPpositive fiber densities inside the corneal epithelium of P2 and adult TRPM8EGFPf+ mice (n = 7 and 5 mice, respectively). The EGFPpositive fiber densities inside the dura of P2 and adult TRPM8EGFPf+ mice are also plotted (similar data as in 5a). p 0.01, p 0.001, twoway ANOVA with post hoc Bonferroni test. ###p 0.001, compared with the P2 dura group. c Percentage adjust of EGFPpositive axon density from P2 to adulthood in the cornea and dura of TRPM8EGFPf+ mice (identical mice as in b). The percentage modify is calculated as (adultdensity – P2density)P2density 100. p 0.001, twotailed ttest.Ren et al. Mol Pain (2015) 11: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)600 500 400 300 200 100 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 region (inside trigeminal V1 dermatome) in 20 min bins in response to dural application of vehicle or IM in adult male mice (n = 12 and 9, respectively). Na e mice (n = six) have been habituated for the test room and recording cage as mice in other groups but have been not subjected to anesthesia exposure, surgery or drug application. b Total duration of nocifensive behavior throughout the 120 min recording period in mice that received dural application of vehicle or IM (exact same mice as in a, p 0.001, twotailed ttest). c Dural application of ()menthol (2.8 mM in 20 ) reduces the duration of automobile and IMinduced nocifensive behavior (n = 6 mice in each group; p 0.001, twoway ANOVA overall effect, p 0.01, p 0.001, post hoc Bonferroni test involving person groups). Co application of menthol and TRPM8 antagonist AMTB (2.8 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, MPP MedChemExpress amongst IM and IM+ AMTB groups, n = 6 and 3 mice, respectively).recorded the duration of nocifensive behavior. Preceding studies show that topical application of 1 mM (-)-menthol produces analgesic effects exclusively.