Table 1 Statistical analyses

Figure 1

 1c (Acetone; injected vs non-injected paw)

Categorical

Non-parametric Wilcoxon matched-pairs signed rank test

vehicle predrug = 9 mice

Wilcoxon matched-pairs signed rank test: DMSO non-injected paw vs. DMSO injected paw pre-drug, p = 0039

 1c (Acetone; injected paw max. effect vs uninjected paw)

Categorical

Non-parametric Mann–Whitney multiple comparisons test, Holm-Šídák method

vehicle uninjected paw day 7 = 9 mice; 4R 15 mg/kg day 7 = 7 mice

Mann–Whitney test, DMSO non-injected paw day 7 vs. 4R 15 mg/kg day 7 injected paw, adjusted p = 0.0086

 1d (Hargreaves; injected vs non-injected paw)

Normal

Two-way ANOVA followed by Tukey's multiple comparisons test

vehicle (predrug, 2–5 h., day 2) = 6 mice; vehicle day 8 = 9 mice; 4R 1 mg/kg (all time points) = 6 mice; 4R 6 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 6 mg/kg day 8 = 8 mice; 4R 15 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 15 mg/kg day 8 = 7 mice

Two-way ANOVA

time: F (3, 172) = 37.71; p < 0.0001

treatment: F (7, 172) = 173.3; p < 0.0001

interaction: F (21, 172) = 6.204; p < 0.0001

posthoc: Tukey’s multiple comparisons test

CFA-injected paw vs uninjected paw in veh conditions: p < 0.0001 (predrug); p < 0.0001 (2–5 h, 6 mg/kg and 15 mg/kg); p < 0.0001 (day 2, 6 mg/kg); p < 0.0001 (day 8, 1 mg/kg); p = 0.2131 (day 8, 6 mg/kg); p = 0.2354 (day 8, 15 mg/kg)

 1d (Hargreaves; injected paw time course)

Normal

Two-way ANOVA followed by Dunnett's multiple comparisons test

vehicle (predrug, 2–5 h., day 2) = 6 mice; vehicle day 8 = 9 mice; 4R 1 mg/kg (all time points) = 6 mice; 4R 6 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 6 mg/kg day 8 = 8 mice; 4R 15 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 15 mg/kg day 8 = 7 mice

Two-way ANOVA

time: F (3, 86) = 59.20; p < 0.0001

treatment: F (3, 86) = 17.87; p < 0.0001

interaction: F (9, 86) = 3.671; p = 0.0006

posthoc: Dunnett’s multiple comparisons test

2–5 h: p = 0.0395 for DMSO vs. 4R 6 mg/kg; p < 0.0001 for DMSO vs. 4R 15 mg/kg

day 2: p = 0.0035 for DMSO vs. 4R 6 mg/kg

day 8: p = 0.0067 for DMSO vs. 4R 1 mg/kg; p < 0.0001 for DMSO vs. 4R 6 mg/kg and DMSO vs. 4R 15 mg/kg

 1d (Hargreaves; non-injected paw time course)

Normal

Two-way ANOVA

vehicle (predrug, 2–5 h., day 2) = 6 mice; vehicle day 8 = 9 mice; 4R 1 mg/kg (all time points) = 6 mice; 4R 6 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 6 mg/kg day 8 = 8 mice; 4R 15 mg/kg (predrug, 2–5 h., day 2) = 6 mice; 4R 15 mg/kg day 8 = 7 mice

Two-way ANOVA

time: F (3, 86) = 2.964; p = 0.0366

treatment: F (3, 86) = 1.450; p = 0.2338

interaction: F (9, 86) = 0.4422; p = 0.9083

 1e (von Frey; injected vs non-injected paw)

Normal

Two-way ANOVA followed by Tukey's multiple comparisons test

vehicle (predrug, 2–5 h.) = 6 mice; vehicle day 2 = 4 mice; vehicle day 8 = 7 mice; 4R 1 mg/kg (predrug, 2–5 h) = 6 mice; 4R 1 mg/kg (day 2, day 8) = 5 mice; 4R 6 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 6 mg/kg day 2 = 5 mice; 4R 15 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 15 mg/kg day 2 = 4 mice

Two-way ANOVA

time: F (3, 148) = 2.378; p = 0.0722

drug treatments: F (7, 148) = 121.8; p < 0.0001

interaction: F (21, 148) = 0.7112; p = 0.8167

posthoc: Tukey’s multiple comparisons test

CFA-injected paw vs uninjected paw in veh conditions; p < 0.0001 (predrug); p < 0.0001 (2–5 h, 1 mg/kg and 15 mg/kg); p < 0.0001 (day 2, 1 mg/kg, 6 mg/kg)

 1e (von Frey; injected paw time course)

Normal

Two-way ANOVA followed by Dunnett's multiple comparisons test

vehicle (predrug, 2–5 h.) = 6 mice; vehicle day 2 = 4 mice; vehicle day 8 = 7 mice; 4R 1 mg/kg (predrug, 2–5 h) = 6 mice; 4R 1 mg/kg (day 2, day 8) = 5 mice; 4R 6 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 6 mg/kg day 2 = 5 mice; 4R 15 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 15 mg/kg day 2 = 4 mice

Two-way ANOVA

time: F (3, 74) = 4.861; p = 0.0038

drug treatments: F (3, 74) = 4.891; p = 0.0037

interaction: F (9, 74) = 1.062; p = 0.4008

posthoc: Dunnett’s multiple comparisons test

2–5 h: p = 0.0403 for DMSO vs. 4R 1 mg/kg; p = 0.0476 for DMSO vs. 4R 15 mg/kg

day 2: p = 0.0487 for DMSO vs. 4R 1 mg/kg; p = 0.0479 for DMSO vs. 4R 6 mg/kg day 8: p = 0.0067 for DMSO vs. 4R 1 mg/kg; p < 0.0001 for DMSO vs. 4R 6 mg/kg and DMSO vs. 4R 15 mg/kg

 1e (von Frey; non-injected paw time course)

Normal

Two-way ANOVA

vehicle (predrug, 2–5 h.) = 6 mice; vehicle day 2 = 4 mice; vehicle day 8 = 7 mice; 4R 1 mg/kg (predrug, 2–5 h) = 6 mice; 4R 1 mg/kg (day 2, day 8) = 5 mice; 4R 6 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 6 mg/kg day 2 = 5 mice; 4R 15 mg/kg (predrug, 2–5 h., day 8) = 6 mice; 4R 15 mg/kg day 2 = 4 mice

Two-way ANOVA

time: F (3, 74) = 0.7107; p = 0.5487

treatment: F (3, 74) = 0.5522; p = 0.6483

interaction: F (9, 74) = 0.7197; p = 0.6892

Figure 2

 2b (Acetone; injected vs. non-injected paw; day 2)

Categorical

Non-parametric Mann–Whitney multiple comparisons test, Holm-Šídák method

vehicle and 4R 15 mg/kg = 6 mice

Mann–Whitney multiple comparisons test, Holm-Šídák method; DMSO injected paw vs. DMSO uninjected paw: adjusted p = 0.0043; DMSO uninjected vs 4R 15 mg/kg in injected paw: adjusted p = 0.0152

 2b (Acetone; veh vs. 4R; day 2)

Categorical

Non-parametric Mann–Whitney multiple comparisons test, Holm-Šídák method

vehicle and 4R 15 mg/kg = 6 mice

Mann–Whitney multiple comparisons test, Holm-Šídák method; DMSO vs. 4R 15 mg/kg in injected paw: adjusted p = 0.0258

 2c (Hargreaves; injected vs non-injected paw; day 2)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 384.3; p < 0.0001

treatment: F (1, 20) = 38.57; p < 0.0001

interaction:F (1, 20) = 26.88; p < 0.0001

posthoc: Šídák’s multiple comparisons test

adjusted p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions;

 2c (Hargreaves; 4R injected vs. DMSO non-injected paw; day 2)

Normal

Two-way ANOVA followed by Tukey's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 384.3; p < 0.0001

treatment: F (1, 20) = 38.57; p < 0.0001

interaction:F (1, 20) = 26.88; p < 0.0001

posthoc: Tukeys multiple comparisons test

adjusted p < 0.0001 for 4R 15 mg/kg CFA-injected paw vs uninjected paw in veh conditions

 2c (Hargreaves;veh vs 4R; day 2)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 384.3; p < 0.0001

treatment: F (1, 20) = 38.57; p < 0.0001

interaction: F (1, 20) = 26.88; p < 0.0001

posthoc: Šídák’s multiple comparisons test

p < 0.0001 for DMSO vs. 4R 15 mg/kg in injected paw

p = 0.7260 for DMSO vs. 4R 15 mg/kg in non-injected paw

 2d (von Frey; injected vs non-injected paw; day 2)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 277.2, p < 0.0001

treatment: F (1, 20) = 1.213; p = 0.2838

interaction:F (1, 20) = 1.213; p = 0.2838

posthoc: Šídák’s multiple comparisons test

p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions

 2d (von Frey; veh vs 4R; day 2)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side:F (1, 20) = 277.2; p < 0.0001

treatment: F (1, 20) = 1.213; p = 0.2838

interaction: F (1, 20) = 1.213; p = 0.2838

posthoc: Šídák’s multiple comparisons test

p = 0.2518 for DMSO vs. 4R 15 mg/kg in injected paw

p > 0.9999 for DMSO vs. 4R 15 mg/kg in non-injected paw

 2e (Acetone; injected vs non-injected paw; day 7)

Categorical

Non-parametric Mann–Whitney multiple comparisons test, Holm-Šídák method

vehicle and 4R 15 mg/kg = 6 mice

Mann–Whitney multiple comparisons test, Holm-Šídák method; DMSO injected paw vs. DMSO uninjected paw: adjusted p = 0.0043, DMSO uninjected vs 4R 15 mg/kg in injected paw: adjusted p = 0.0043

 2f (Hargreaves; injected vs non-injected paw; day 7)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 42.74; p < 0.0001

treatment: F (1, 20) = 7.807; p = 0.0112

interaction:F (1, 20) = 5.150; p = 0.0345

posthoc: Šídák’s multiple comparisons test

adjusted p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions,

 2f (Hargreaves; 4R injected vs. DMSO non-injected paw; day 7)

Normal

Two-way ANOVA followed by Tukey's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 42.72; p < 0.0001

treatment: F (1, 20) = 7.807; p = 0.0112

interaction:F (1, 20) = 5.150; p = 0.0345

posthoc: Tukeys multiple comparisons test

adjusted p = 0.0679 for 4R 15 mg/kg CFA-injected paw vs uninjected paw in veh conditions

 2f (Hargreaves;veh vs 4R; day 7)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 42.74; p < 0.0001

treatment: F (1, 20) = 7.807; p = 0.0112

interaction: F (1, 20) = 5.150; p = 0.0345

posthoc: Šídák’s multiple comparisons test

p = 0.0037 for DMSO vs. 4R 15 mg/kg in injected paw

p = 0.9185 for DMSO vs. 4R 15 mg/kg in non-injected paw

 2 g (von Frey; injected vs non-injected paw; day 7)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 74.43; p < 0.0001

treatment: F (1, 20) = 0.8013; p = 0.3814

interaction: F (1, 20) = 0.01463; p = 0.9049

posthoc: Šídák’s multiple comparisons test

p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions

 2g (von Frey; veh vs 4R; day 7)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way ANOVA

paw side: F (1, 20) = 74.43; p < 0.0001

treatment: F (1, 20) = 0.8013; p = 0.3814

interaction: F (1, 20) = 0.01463; p = 0.9049

posthoc: Šídák’s multiple comparisons test

p = 0.7304 for DMSO vs. 4R 15 mg/kg in injected paw

p = 0.8320 for DMSO vs. 4R 15 mg/kg in non-injected paw

Figure 3

 3b (thickness of injected vs non-injected paw, male)

Normal

Two-way ANOVA followed by Tukey's multiple comparisons test

vehicle (predrug, 2–5 h., day 2) = 16 mice; vehicle (day 3, day 7) = 12 mice; vehicle day 8 = 9; 4R 1 mg/kg (predrug, 2–5 h., day 2) = 14 mice; 4R 1 mg/kg (day 3, day 7) = 12 mice; 4R 1 mg/kg day 8 = 6 mice; 4R 6 mg/kg (predrug, 2–5 h., day 2) = 16 mice; 4R 6 mg/kg (day 3, day 7) = 12 mice; 4R 6 mg/kg day 8 = 8 mice; 4R 15 mg/kg (predrug, 2–5 h., day 2) = 14 mice; 4R 15 mg/kg (day 3, day 7) = 12 mice; 4R 15 mg/kg day 8 = 7 mice

Two-way ANOVA

time: F (5, 564) = 45.20; p < 0.0001

drug treatments: F (7, 564) = 447.0; p < 0.0001

interaction: F (35, 564) = 8.479; p < 0.0001

posthoc: Tukey’s multiple comparisons test

CFA-injected paw vs uninjected paw in veh conditions; p < 0.0001 (predrug); p < 0.0001 (day 7, 4R 6 mg/kg)

 3b (injected paw thickness time course, male)

Normal

Two-way ANOVA followed by Dunnett's multiple comparisons test

vehicle (predrug, 2–5 h., day 2) = 16 mice; vehicle (day 3, day 7) = 12 mice; vehicle day 8 = 9; 4R 1 mg/kg (predrug, 2–5 h., day 2) = 14 mice; 4R 1 mg/kg (day 3, day 7) = 12 mice; 4R 1 mg/kg day 8 = 6 mice; 4R 6 mg/kg (predrug, 2–5 h., day 2) = 16 mice; 4R 6 mg/kg (day 3, day 7) = 12 mice; 4R 6 mg/kg day 8 = 8 mice; 4R 15 mg/kg (predrug, 2–5 h., day 2) = 14 mice; 4R 15 mg/kg (day 3, day 7) = 12 mice; 4R 15 mg/kg day 8 = 7 mice

Two-way ANOVA

time: F (5, 282) = 50.75; p < 0.0001

drug treatments: F (3, 282) = 12.64; p < 0.0001

interaction:F (15, 282) = 1.301; p = 0.2007

posthoc: Dunnett’s multiple comparisons test

day 2: p = 0.0212 for DMSO vs. 4R 15 mg/kg

day 3: p = 0.0061 for DMSO vs. 4R 6 mg/kg; p = 0.0165 for DMSO vs. 4R 15 mg/kg

day 7: p = 0.0006 for DMSO vs. 4R 6 mg/kg; p = 0.0061 for DMSO vs. 4R 15 mg/kg

day 8: p = 0.0172 for DMSO vs. 4R 6 mg/kg; p = 0.0152 for DMSO vs. 4R 15 mg/kg

 3c (thickness of injected vs non-injected paw, female)

Normal

Two-way RM ANOVA followed by Tukey’s multiple comparisons test

vehicle and 4R 15 mg/kg = 6 mice

Two-way RM ANOVA

time: F (1.448, 28.95) = 88.41; p < 0.0001

drug treatments: F (3, 20) = 520.4; p < 0.0001

time X drug treatments: F (15, 100) = 33.57; p < 0.0001

posthoc: Tukey's multiple comparisons test

pre-drug: p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions

time: F (5, 60) = 70.27; p < 0.0001

drug treatments: F (1, 60) = 0.5000; p = 0.4822

interaction: F (5, 60) = 2.675; p = 0.0301

 3f (injected paw glabrous skin thickness)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

Vehicle—male = 6 mice

Two-way ANOVA

Vehicle—female = 6 mice

drug treatment: F (1, 19) = 4.647; p = 0.0441

4R—male = 5 mice

sex: F (1, 19) = 0.294; p = 0.5936

4R—female = 6 mice

interaction: F (1, 19) = 0.056; p = 0.8140

posthoc: Šídák’s multiple comparisons test

p = 0.3617 for Males Vehicle vs. Males 4R

p = 0.1881 for Females Vehicle vs. Females 4R

 3g (injected paw subdermal cell count)

Normal

Two-way ANOVA followed by Šídák's multiple comparisons test

Vehicle—male = 6 mice

Two-way ANOVA

Vehicle—female = 6 mice

drug treatment: F (1, 19) = 7.495; p = 0.0131

4R—male = 5 mice

sex: F (1, 19) = 0.4968; p = 0.48985

4R—female = 6 mice

interaction: F (1, 19) = 7.073; p = 0.0155

posthoc: Šídák’s multiple comparisons test

p = 0.0028 for Males Vehicle vs. Male 4R

p = 0.9980 for Females Vehicle vs. Females 4R

p = 0.0489 for Males Vehicle vs. Females Vehicle

p = 0.3484 for Males 4R vs. Females 4R

Figure 4

 4b (Acetone)

Categorical

Non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparisons test

MLA + DMSO, MLA + 4R, Saline + 4R and Saline + DMSO = 6 mice

Kruskal–Wallis test: p = 0.0076; posthoc: Dunn’s mutiple comparisons test: Saline + 4R vs. MLA + 4R p = 0.0041; Saline + DMSO vs. MLA + DMSO p = 0.7503;

 4c (Hargreaves)

Non-normal

Non-parametric Kruskal–Wallis test followed by Dunn's multiple comparisons test

MLA + DMSO, MLA + 4R, Saline + 4R and Saline + DMSO = 6 mice

Kruskal–Wallis test: p = 0.0005; posthoc: Dunn's mutiple comparisons test: Saline + 4R vs. MLA + 4R p = 0.0058; Saline + DMSO vs. MLA + DMSO p = 0.0825;

 4d (thickness of injected vs non-injected paw, male)

Normal

Two-way RM ANOVA followed by Tukey's multiple comparisons test

MLA + DMSO = 6 mice, MLA + 4R = 6 mice, Saline + 4R = 6 mice and Saline + DMSO = 3–6 mice

Two-way ANOVA

time: F (1.561, 57.76) = 37.40; p < 0.0001

drug treatments: F (7, 37) = 290.5; p < 0.0001

interaction: F (35, 185) = 7.757; p < 0.0001

posthoc: Tukey’s multiple comparisons test

pre-drug: p < 0.0001 for CFA-injected paw vs uninjected paw in veh conditions

 4d (injected paw thickness time course, male)

Normal

Two-way RM ANOVA followed by Tukey’s multiple comparisons test

MLA + DMSO = 6 mice, MLA + 4R = 6 mice, Saline + 4R = 6 mice and Saline + DMSO = 3–6 mice

Two-way ANOVA

time: F (1.403, 28.06) = 52.55; p < 0.0001

drug treatments: F (3, 20) = 2.550; p = 0.0846

interaction: F (15, 100) = 1.651; p = 0.0737

posthoc: Tukey’s multiple comparisons test

day 2: p = 0.0076 for Saline + 4R vs. Saline + DMSO

day 3: p = 0.0026 for Saline + 4R vs. Saline + DMSO

day 7: p = 0.0200 for Saline + 4R vs. Saline + DMSO; p = 0.0069 for MLA + 4R vs. Saline + 4R

day 8: p = 0.0116 for Saline + 4R vs. Saline + DMSO; p = 0.0043 for MLA + 4R vs. Saline + 4R

Figure 5

 5c (injected paw subdermal α7^tdT cell count)

Normal

Two-way ANOVA

Vehicle—male = 5 mice

Two-way ANOVA

Vehicle—female = 4 mice

drug treatments: F(1, 13) = 0.001727; p = 0.9675

4R—male = 4 mice

4R—female = 4 mice

sex: F(1, 13) = 0.008159; p = 0.9294

interaction: F(1, 13) = 0.6627; p = 0.4303

 5d (injected paw subdermal Iba-1⁺ cell count

Normal

Two-way ANOVA

Vehicle—male = 4 mice

Two-way ANOVA

Vehicle—female = 4 mice

drug treatments: F(1, 12) = 0.2515; p = 0.6251

4R—male = 4 mice

sex: F(1, 12) = 0.02600; p = 0.6251

4R—female = 4 mice

interaction: F(1, 12) = 0.8920; p = 0.3636

 5e (injected paw subdermal colocalized cell count

Normal

Two-way ANOVA

Vehicle—male = 5 mice

Two-way ANOVA

Vehicle—female = 4 mice

drug treatments: F(1, 13) = 0.6376; p = 0.4389

4R—male = 4 mice

sex: F(1, 12) = 0.05774; p = 0.8138

4R—female = 4 mice

interaction: F(1, 12) = 0.5835; p = 0.4586

 5h (injected paw subdermal α7^tdT cell count

Normal

Paired t-test

CFA-injected—male = 4 mice

Paired t-test (F4/80)

uninjected—male = 4 mice

p value: 0.5122

CFA-injected—female = 4 mice

t, df: t = 0.6904, df = 7

uninjected—female = 4 mice

Paired t-test (CD3 +)

p value: 0.9088

t, df: t = 0.1188, df = 7