Figure 4.

Nociceptive responses to oxylipins in control and KO mice. The cheek wipes were measured in the first (a) 5 minutes and (b) 30 minutes. Significance was determined using the Kruskal‒Wallis test with Dunn’s test for multiple comparisons: 9,13-EHL versus vehicle, 9-HODE, and 2-hydroxy-5,6-epoxy-hept-3-(E)-ene is not significant; other P-values when compared with 9,13-EHL are as follows: versus PGE₂ ∗∗P = 0.059, versus 2,2M-9,13-EHL ∗P = 0.0230, versus 13M-9,13-EHL ∗∗P = 0.0023, versus 2,2,13M-9,13-EHL ∗P = 0.023, and versus 9,10,13-THL ∗P = 0.0282. The same statistical tests were used to compare 9,10,13-THL with vehicle ∗∗P = 0.0033, with 9-HODE (not significant), versus PGE₂ (not significant), versus 2,2M-9,10,13-THL ∗P = 0.0282, versus 2,2,13M-9,10,13-THL (not significant), and versus 2,5,6-trihydroxy-hept-3-(E)-ene (not significant). (c) quantification of ipsilaterally directed cheek scratching bouts after injection of vehicle (1% ethanol in 1X PBS [v/v]) and various mediators (100 μg/20 μl i.d.). No significant differences were found in the number of ipsilaterally cheek-directed scratching bouts using the same Kruskal‒Wallis test with Dunn’s correction for multiple comparisons. (d) Total time spent on paw-directed nociceptive behavior response after intraplantar injection of over 5 minutes. The significance was determined using a Kruskal‒Wallis test with a Dunn’s test for multiple comparisons: vehicle versus 9-HODE ∗∗P = 0.0090, vehicle versus PGE₂ ∗∗P = 0.0013, and vehicle versus 9,10,13-THL ∗∗P = 0.0029. e) The total number of wipes quantified after vehicle or 100 μg/20 μl 9,10,13-THL (i.d.) in WT, Trpa1-KO, and Trpv1-KO mice. Significance was determined using a one-way ANOVA with the Holm-Šídák correction for multiple comparisons: vehicle versus Trpa1-KO (not significant), vehicle versus Trpv1 KO ∗∗∗P = 0.0004, vehicle versus WT ∗∗∗∗P < 0.0001, Trpa1 KO versus Trpv1 KO ∗∗∗∗P < 0.0001, Trpa1 KO versus WT ∗∗∗P = 0.0007, and Trpv1 KO versus WT ∗∗∗P = 0.0007. (f) Time kinetics of behavior of 9,10,13-THL after injection in 5-minute intervals. Significance was determined using the same parameters as panel e: for the 5-minute bin, no significant differences were found between the number of ipsilaterally cheek-directed wipes in the vehicle-treated mice and the Trpa1-KO mice treated with 9,10,13-THL. Similarly, no significant differences were found between the 9,10,13-THL‒treated Trpa1-KO and 9,10,13-THL‒treated Trpv1-KO mice. The other significance values are as follows: vehicle versus WT ∗∗P = 0.0040, vehicle versus Trpv1 KO ∗P = 0.0104, WT versus Trpa1 KO ∗∗P = 0.0035, and WT versus Trpv1 KO ∗P = 0.0104. Data are presented as mean ± SD, and each data point in panels a–e corresponds to one biological replicate. n ≥ 5. 13M-9,13-EHL, 13-methyl, 9,10-epoxy octadecenoate; 2,2,13M-9,10,13-THL, 2,2,13-methyl-9,10,13-hydroxy octadecenoate; 2,2M-9,10,13-THL, 2,2-methyl-9,10,13-hydroxy octadecenoate; 2,2M-9,13-EHL, 2,2, methyl-13-hydroxy-9,10-epoxy octadecenoate; 2,2,13M-9,13-EHL, 2,2,13-methyl-9,10-epoxy octadecenoate; 9,10,13-THL, 9,10,13-trihydroxy-octadecenoate; 9,13-EHL, 13-hydroxy-9,10-epoxy octadecenoate; HODE, hydroxyoctadecenoate; i.d., intradermally; KO, knockout; min, minute; ns, not significant; PGE₂, prostaglandin E₂; WT, wild type.