H₂O₂-induced nociceptive behavior in rats as total time spent flinching, lifting and licking of the hind leg.

<p>(A) Spontaneous nociceptive behavior after intramuscular (n = 6) or subcutaneous (n = 5) injection of H₂O₂ (100 mM, 0.6 ml), or intramuscular injection of synthetic interstitial fluid (0.6 ml) (n = 6). * P = 0.0004 compared with control group, † P = 0.0010 compared with subcutaneous H₂O₂ injection group by one-way ANOVA followed by post-hoc Bonferroni’s test. (B) Effects of local pre-injection of a TRPA1 antagonist (HC-030031; 50 mM, 0.3 ml, n = 6) on nociceptive behavior caused by intramuscular injection of H₂O₂ (100 mM, 0.3 ml, n = 7) vs. vehicle group (n = 7). * P < 0.0001 compared with vehicle + PBS injection group, † P < 0.0001 compared with HC-030031 + H₂O₂ injection group by one-way ANOVA followed by post-hoc Bonferroni’s test. All data are expressed as means ± SEM.</p

Effect of intraperitoneal (i.p.) administration of HC-030031 on pain behaviors of rats after skin + deep tissue incision.

<p>(A) Guarding pain behavior. The results are presented as mean and standard error of the mean (SEM) for eight rats in each group. Two-way ANOVA with repeated measures on one factor (interaction factor: F_{24, 210} = 1.64, P = 0.0360) followed by Bonferroni’s post hoc test for comparing the mean cumulative pain score at each time point among groups. (B) Withdrawal threshold to punctate stimuli applied to the hind paw. The results are presented as median with range for six rats in each group. Non-parametric Friedman’s test (Fr = 17.02, P = 0.0019) followed by Kruskal-Wallis test with Dunn post hoc test for between-group comparisons at each time point. (C) Withdrawal latency to heat stimulation. The results are presented as mean and SEM. Two-way ANOVA with repeated measures on one factor (interaction factor: F_{24, 150} = 1.163, P = 0.2855) followed by Bonferroni’s post hoc test for comparing the mean withdrawal latency at each time point among groups. * P < 0.05, ** P < 0.01, † P < 0.001 compared with the vehicle group at each time point. POD = postoperative day.</p

<i>In vivo</i> reactive oxygen species (ROS)-imaging with L-012 after incision in rats.

<p>(A) Examples of <i>in vivo</i> imaging after gastrocnemius muscle incision. (B) Average luminescence intensity in <i>in vivo</i> ROS-imaging on gastrocnemius muscle incision, gastrocnemius incision with catalase (1,000–2,500 IU), skin-only incision, and sham. The results are presented as mean and SEM for 6 rats in each group. * P < 0.0001 compared with sham, † P < 0.0001 compared with gastrocnemius incision with catalase, ‡ P < 0.0001 compared with skin-only incision, # P < 0.0001 compared with sham, § P = 0.0159 compared with gastrocnemius incision with catalase, & P = 0.0490 compared with skin-only incision, ¶ P = 0.0166 compared with sham. Two-way ANOVA (interaction factor: F_{6, 30} = 22.56, P < 0.0001) followed by Bonferroni's post hoc tests.</p

Effects of the incision of skin and muscle on tissue hydrogen peroxide (H₂O₂) levels in rats.

<p>(A) H₂O₂ content after incision in gastrocnemius muscle using the Amplex^® Red Hydrogen Peroxide assay kit. The results are presented as mean and SEM for 6 rats in each group. Two-way ANOVA with repeated measures on one factor (interaction factor: F_{2, 15} = 2.328, P = 0.1317, Time factor: F_{2, 15} = 3.890, P = 0.0436, Group factor: F_{1, 15} = 22.58, P = 0.0003) followed by Bonferroni’s post hoc test. * P < 0.0001 compared with non-incised muscle on POD 0, † P = 0.0074 compared with non-incised muscle on POD 1. (B) H₂O₂ content after incision of skin overlying the gastrocnemius muscle. The results are presented as mean and SEM for 6 rats in each group. Two-way ANOVA with repeated measures on one factor (interaction factor: F_{2, 15} = 0.5907, P = 0.5663, Time factor: F_{2, 15} = 0.2134, P = 0.8103, Group factor: F_{1, 15} = 31.71, P < 0.0001) followed by Bonferroni’s post hoc test. # P = 0.0105 compared with incised skin on POD 0, ‡ P = 0.0373 compared with incised skin on POD 1.</p

Proportions and the average conduction velocities (CVs) of the different classes of cutaneous afferents.

<p>Values for CV are means ± SEM. *<i>p</i> < 0.05 <i>vs</i>. WT by unpaired t-test.</p><p>Abbreviation: AM, A-mechanonociceptor; DH, D-hair receptor; RA, Rapidly adapting low-threshold mechanoreceptor; SA, Slowly adapting low-threshold mechanoreceptor; WT, wild-type C57BL/6J control mice; TKO, <i>ASIC</i> triple-knockout mice; NS, not significant.</p

Proportions of the different classes and conduction velocity distribution of mechanosensitive afferents.

<p><b><i>A</i></b>, Percentage occurrence of C-fibers, A-mechanonociceptors (AMs), D-hair receptors (DHs), rapidly adapting (RAs) and slowly adapting low-threshold mechanoreceptors (SAs) identified in <i>ASIC</i> triple-knockout (TKO) and wild-type (WT) mice. ^&One slowly adapting Aδ-unit from a WT mouse could not be classified as one of five classes, because it had mechanical threshold of less than 1mN. <b><i>B</i></b>, Conduction velocity distribution histogram. <b><i>C</i></b>, Percentage of AM that conducted in the Aδ and Aβ conduction velocity range. <b><i>D</i></b>, <b><i>E</i></b>, Percentage of Aδ- (<b><i>D</i></b>) and Aβ-fibers (<b><i>E</i></b>) that were classified as AM, DH, SA or RA.</p

Adaptation properties of cutaneous primary afferents in <i>ASIC</i> triple-knockout (TKO) and wild-type (WT) mice.

<p><b><i>A</i></b>, AM from <i>ASIC</i> TKO mice showed increased firing initially and throughout the duration of sustained, 80-mN stimulus (<i>P</i>  =  0.001 between genotypes by two-way ANOVA with repeated measures on one factor. *<i>P</i> < 0.05; **<i>P</i> < 0.01; †<i>P</i> < 0.001 <i>vs</i>. WT by unpaired t-test). <b><i>B</i></b>, When the action potentials in each 0.3-s bin were normalized to total spikes generated during the entire duration of stimulus, there was no significant difference between the two genotypes. <b><i>C</i></b>, <b><i>D</i></b>, When AM fibers were subclassified based on conduction velocity, not Aβ-AM (<b><i>C</i></b>) but Aδ-AM (<b><i>D</i></b>) from <i>ASIC</i> TKO mice showed a significantly increased firing initially and throughout the duration of the stimulus (<i>p</i>  =  0.005 between genotypes by two-way ANOVA with repeated measures on one factor. *<i>p</i> < 0.05; **<i>p</i> < 0.01 <i>vs</i>. WT by unpaired t-test). <b><i>E-H</i></b>, Average action potentials in 0.3-s bins during a sustained force stimulus in C-fibers (<b><i>E</i></b>), D-hair receptors (DHs) (<b><i>F</i></b>), rapidly adapting (RAs) (<b><i>G</i></b>), and slowly adapting low-threshold mechanoreceptors (SAs) (<b><i>H</i></b>). Data are presented as mean ± SEM.</p

Fiber classification of mouse cutaneous mechanosensitive afferents.

<p>Fiber classification of mouse cutaneous mechanosensitive afferents.</p

Mechanosensitivity of cutaneous Aδ-mechanonociceptors (Aδ-AM ) in <i>ASIC</i> triple-knockout (TKO) and wild-type (WT) mice.

<p> Aδ-AM fibers were further subdivided into two groups based on conduction velocity (CV) range (CV ≤ 2.0 <i>vs</i>. CV > 2.0). <b><i>A</i></b>, Conduction velocity distribution histogram of AM fibers. <b><i>B</i></b>, Mechanical stimulus-response function of Aδ-AM from WT mice. Only one fiber conducted slower than 2 m/s (▪). <b><i>C</i></b>, Mechanical stimulus-response function of Aδ-AM from <i>ASIC</i> TKO mice. Fibers that conducted faster than 2 m/s (<i>n</i>  =  17, ▵) showed a tendency toward enhanced mechanosensitivity, compared to those that conducted slower than 2 m/s (<i>n</i>  =  8, □) (<i>p</i>  =  0.090 between CV groups by two-way ANOVA with repeated measures on one factor). Data are presented as mean ± SEM.</p

Mechanosensitivity of cutaneous primary afferents in <i>ASIC</i> triple-knockout (TKO) and wild-type (WT) mice.

<p><b><i>A</i></b>, Sample recording traces showing responses of A-mechanonociceptors (AM) from WT and <i>ASIC</i> TKO mice to mechanical stimuli. The <i>upper, middle and lower panels</i> show the digitized oscilloscope tracing, the spike density histograms (bin width  =  1 s), and the force stimuli applied, respectively. CV  =  conduction velocity. <b><i>B</i></b>, Stimulus-response function of AM from <i>ASIC</i> TKO (<i>n</i>  =  40, ○) <i>vs</i>. WT (<i>n</i>  =  35, •), showing enhanced mechanosensitivity in <i>ASIC</i> TKO mice (<i>p</i>  =  0.006 between genotypes by two-way ANOVA with repeated measures on one factor. **<i>p</i> < 0.01; †<i>p</i> < 0.001 <i>vs</i>. WT by unpaired t-test). <b><i>C</i></b>, <b><i>D</i></b>, When AM fibers were subclassified based on CV, not Aβ-AM (<b><i>C</i></b>) but Aδ-AM (<b><i>D</i></b>) showed a significant increase in the stimulus-response function in <i>ASIC</i> TKO mice (<i>p</i>  =  0.028 between genotypes by two-way ANOVA with repeated measures on one factor. *<i>p</i> < 0.05; **<i>p</i> < 0.01 <i>vs</i>. WT by unpaired t-test). <b><i>E-H</i></b>, Stimulus-response function of C-fibers (<b><i>E</i></b>), D-hair receptors (DHs) (<b><i>F</i></b>), rapidly adapting (RAs) (<b><i>G</i></b>), and slowly adapting low-threshold mechanoreceptors (SAs) (<b><i>H</i></b>). Data are presented as mean ± SEM.</p