An improved conflict avoidance assay reveals modality-specific differences in pain hypersensitivity across sexes

Abstract

Abnormal encoding of somatosensory modalities (ie, mechanical, cold, and heat) are a critical part of pathological pain states. Detailed phenotyping of patients' responses to these modalities have raised hopes that analgesic treatments could one day be tailored to a patient's phenotype. Such precise treatment would require a profound understanding of the underlying mechanisms of specific pain phenotypes at molecular, cellular, and circuitry levels. Although preclinical pain models have helped in that regard, the lack of a unified assay quantifying detailed mechanical, cold, and heat pain responses on the same scale precludes comparing how analgesic compounds act on different sensory phenotypes. The conflict avoidance assay is promising in that regard, but testing conditions require validation for its use with multiple modalities. In this study, we improve upon the conflict avoidance assay to provide a validated and detailed assessment of all 3 modalities within the same animal, in mice. We first optimized testing conditions to minimize the necessary amount of training and to reduce sex differences in performances. We then tested what range of stimuli produce dynamic stimulus-response relationships for different outcome measures in naive mice. We finally used this assay to show that nerve injury produces modality-specific sex differences in pain behavior. Our improved assay opens new avenues to study the basis of modality-specific abnormalities in pain behavior.

Keywords: Cold; Conflict avoidance; Cuff; Female; Heat; Mechanical; Modality; Mouse; Neuropathic pain; Operant pain behavior; Preclinical; Preclinical pain models; Sex difference; Thermal; Translation; c57bl/6.

Figure 1.

Validation of the effect of aversion to the initial compartment on training performances. (A) Diagram of the original and modified apparatus with measures of interest. (B) Diagram of the training protocol. (C and D) Percentage of the time spent in the initial compartment in the original and modified apparatus (n = 12 each), with and without light for (C) C57BL/6J mice (compartment F_1,22 = 20.11, P = 0.0002, RM 2-way ANOVA) and (D) CD-1 mice (Compartment x light, F_1,22 = 32.30, P <0.0001, RM 2-way ANOVA). Sex-separated values for CD1 are provided (D, right panel). Male mice are represented in black, and female mice in grey. (E) Difference between light off and on conditions for the modified compartment in both strains tested. (F and G) Comparison of escape and return latencies over the training procedure in the original (F), and modified (G) apparatus. Escape (H) and return (I) latencies of male and female mice trained in the original apparatus (Escape F_1,18 = 3.739, P = 0.07; Return F_1,18 = 10.73, P = 0.004, RM 2-way ANOVA). (J and K) Similar comparison for the modified apparatus (Escape F_1,14 = 2.713, P = 0.1; Return F_1,14 = 0.07963, P = 0.8, RM 2-way ANOVA). Data are shown as mean ± SEM. Differences between the groups are represented as asterisks (*), results from post hoc tests are represented as hashes (#), and results from the Mann–Whitney test are represented as a section sign (§). **P < 0.01, ^#P < 0.05, ^§§§P < 0.001, ^####P < 0.0001, ^§§§P < 0.001, ^§§§§ P < 0.0001, ns, not significant. ANOVA, analysis of variance; RM, repeated measures.

Figure 2.

Aversive response to mechanical probes of different sizes in naive male and female mice. (A) Diagram of the testing protocol for all modalities. (B) Microscopic images of nail and pin tips (left and right panel, respectively; scale bar = 500 µm). (C) Escape latency of male mice exposed to nails (open circles) or pins (full circles) (stimulus × probe height F_3,130 = 8.105, P < 0.0001, 2-way ANOVA). (D) Return latency of male mice exposed to nails or pins (probe height F_2.242,79.23 = 6.662, P = 0.001; stimulus F_1,45 = 5.307, P = 0.03, 2-way ANOVA). (E) Escape latency of female mice exposed to nails or pins (stimulus F_1,48 = 3.735, P = 0.06; Probe Height F_{2.470, 112.0} = 10.89, P < 0.0001). (F) Return latency of female mice exposed to nails or pins (stimulus × probe height F_{3, 114} = 3.532, P = 0.02, 2-way ANOVA). Data are shown as mean ± SEM. Differences from baseline for pins are represented as hashes (#). Scale bar = 500um. ^##P < 0.05, ^###P < 0.001, ^####P < 0.0001. ANOVA, analysis of variance.

Figure 3.

Aversive response to various temperatures in naive male and female mice. (A) Escape and (B) return latency of male (blue) and female (light blue) mice in response to cold temperatures (escape sex × temperature F_{3, 96} = 15.45, P < 0.0001; Return Sex × Temperature F_{3, 78} = 2.974, P = 0.04, RM 2-way ANOVA and 2-way ANOVA). (C) Escape and (D) return latency of males (red) and females (pink) in response to hot temperatures (Return Temperature F_{2.674, 75.76} = 18.50, P < 0.0001, RM 2-way ANOVA; Return Sex F_{1, 29} = 0.3699, P = 0.6, RM 2-way ANOVA). Data are shown as mean ± SEM. Differences from baseline are represented as hashes (#) for male mice and section signs (§) for female mice. *P < 0.05, ****P < 0.0001, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001, ^§P < 0.05, ^§§P < 0.01, ^§§§P < 0.001, ns, not significant. ANOVA, analysis of variance, RM, repeated measures.

Figure 4.

Validation of hypersensitivity following surgery using the von Frey test, a classical reflexive method. Paw withdrawal threshold of male and female naive, cuff, and SNI mice. **P < 0.01, ****P < 0.0001. PWT, paw withdrawal threshold; SNI, spared nerve injury.

Figure 5.

Aversive response of male mice to mechanical probes following nerve injury. (A and B) Escape and return latency of nerve-injured male mice in comparison to naive male mice (Escape Injury × Probe height F_{6, 163} = 10.09, P < 0.0001, 2-way ANOVA; Return Injury × Probe height F_{6, 83} = 4.296, P = 0.0008, 2-way ANOVA). (C and D) Percentage of escape and return success of naive and nerve-injured male mice in the presence of mechanical probes. Data are shown as mean ± SEM. Results from post hoc tests comparing naive vs cuffs (#), naive vs SNI (§), and cuff vs SNI (†). Results from Fisher exact test are represented by asterisks (*). *P < 0.05, **P < 0.01, ***P < 0.001, ^####P < 0.0001, ^§P < 0.05, ^§§§P < 0.001, ^§§§§P < 0.0001, ^†P < 0.05. ANOVA, analysis of variance; SNI, spared nerve injury.

Figure 6.

Aversive response of male mice to thermal stimuli following nerve injury. (A and B) Escape and return latency of naive and nerve-injured male mice in response to cold temperatures (Escape Temperature × Injury F_{6, 120} = 2.273, P = 0.04, RM 2-way ANOVA; Return Injury F_{2, 40} = 0.2559, P = 0.8, 2-way ANOVA). (C and D) Percentage of escape and return success of naive and nerve-injured male mice in response to cold temperatures. (E and F) Escape and return latency of naive and nerve-injured male mice in response to hot temperatures (Escape Injury F_2,40 = 0.09868, P = 0.9, RM 2-way ANOVA; Return Injury F_2,38 = 2.109, P = 0.1, 2-way ANOVA). (G and H) Percentage of escape and return success of naive and nerve-injured male mice in response to hot temperatures. Data are shown as mean ± SEM. Results from post hoc tests comparing naive vs SNI are represented as section signs (§). Results from Fisher exact test are represented by asterisks (*). *P < 0.05, ^§P < 0.05. ANOVA, analysis of variance; RM, repeated measures; SNI, spared nerve injury.

Figure 7.

Aversive response of female mice to mechanical probes following nerve injury. (A and B) Escape and return latency of female cuffs and SNIs in comparison to naive female mice (Escape Injury x Probe height F_{6, 163} = 3.837, P = 0.001, 2-way ANOVA; Return Injury × Probe Height F_{6, 123} = 2.158, P = 0.05, 2-way ANOVA). (C and D) Percentage of escape and return success of naive and nerve-injured female mice in response to mechanical probes. Data are shown as mean ± SEM. Results from post hoc tests comparing naive vs cuffs (#), naive vs SNI (§), and cuff vs SNI (†). Results from Fisher exact test are represented by asterisks (*). **P < 0.01, ^#P < 0.05, ^§P < 0.05, ^§§P < 0.01, ^†P < 0.05. ANOVA, analysis of variance; SNI, spared nerve injury.

Figure 8.

Aversive response of female mice to thermal stimuli following nerve injury. (A and B) Escape and return latency of naive, cuff and SNI female mice in response to cold temperatures (Escape Injury × Temperature F_{6, 129} = 10.03, P < 0.0001, RM 2-way ANOVA; Return Injury × Temperature F_{6, 100} = 1.209, P = 0.3, 2-way ANOVA). (C and D) Percentage of escape and return success of naive and nerve-injured females in response to cold temperatures. (E and F) Escape and return latency of naive and nerve-injured female mice in response to hot temperatures (Escape Injury × Temperature F_{6, 129} = 1.205, P = 0.3, RM 2-way ANOVA; Return Injury F_{2, 39} = 0.5396, P = 0.6, 2-way ANOVA). (G and H) Percentage of escape and return success of naive, cuff and SNI female mice in response to hot temperatures. Data are shown as mean ± SEM. Results from post hoc tests comparing naive vs cuffs (#) and naive vs SNI (§). Results from Fisher exact test are represented by asterisks (*). *P < 0.05 **P < 0.01, ^##P < 0.01, ^###P < 0.001, ^§§§§P < 0.0001. ANOVA, analysis of variance; RM, repeated measures; SNI, spared nerve injury.