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

Ation of IM is actually a well-established preclinical model of headache [372]. Initially, we modified the composition of IM and applied it onto the dura of well-habituated adult male mice. The home-cage behavior of mice getting 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 duration of wiping and scratching peaked 400 min soon after IM exposure and gradually subsided (Figure 7a). Mice that received dural IM application exhibited considerably longer duration of wiping and scratching than mice treated with vehicle (Figure 7b, p 0.001, two-tailed t-test), suggesting that meningeal irritation elicits ongoing Bromophenol blue Description nocifensive behavior in adult mice. Subsequent, we co-applied two.eight mM TRPM8 agonist (-)-menthol together with the automobile or IM onto the dura andaPb9 eight 7 six five 4 3 two 1Axon Density (mm-1)Cornea Dura###25change of axon densityAdultcPAdult80 60 40 20 0 -20 -40 -CorneaEGFPf+DuraFigure six Postnatal raise in 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 within 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 information as in 5a). p 0.01, p 0.001, twoway ANOVA with post hoc Bonferroni test. ###p 0.001, compared using the P2 dura group. c Percentage adjust of EGFPpositive axon density from P2 to adulthood within the cornea and dura of TRPM8EGFPf+ mice (very same mice as in b). The percentage adjust 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 Chlortetracycline In stock inhibits meningeal irritationinduced ongoing nocifensive behavior in adult mice. a Time spent on forepaw wiping and hindpaw scratching around the scalp and periorbital region (inside trigeminal V1 dermatome) in 20 min bins in response to dural application of car 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 have been not subjected to anesthesia exposure, surgery or drug application. b Total duration of nocifensive behavior in the course of the 120 min recording period in mice that received dural application of car or IM (similar mice as inside a, p 0.001, twotailed ttest). c Dural application of ()menthol (two.eight mM in 20 ) reduces the duration of automobile and IMinduced nocifensive behavior (n = 6 mice in each and every group; p 0.001, twoway ANOVA overall effect, p 0.01, p 0.001, post hoc Bonferroni test amongst person groups). Co application of menthol and TRPM8 antagonist AMTB (2.eight mM in 20 ) reverses the effect of menthol (n = three mice; p 0.01, p 0.001). AMTB does not alter the duration of IMinduced nocifensive behavior (p = 0.72, among IM and IM+ AMTB groups, n = six and 3 mice, respectively).recorded the duration of nocifensive behavior. Earlier research show that topical application of 1 mM (-)-menthol produces analgesic effects exclusively.