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. 2024 Apr 1:15:100155.
doi: 10.1016/j.ynpai.2024.100155. eCollection 2024 Jan-Jun.

Thermal escape box: A cost-benefit evaluation paradigm for investigating thermosensation and thermal pain

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Thermal escape box: A cost-benefit evaluation paradigm for investigating thermosensation and thermal pain

Jacquelyn R Dayton et al. Neurobiol Pain. .

Abstract

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8-/- mice and in male Trpv1-/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain.

Keywords: Cost-benefit analysis; Decision-based behavior; Nociception; Thermal allodynia; Thermal hyperalgesia; Thermosensation.

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Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Theanne N. Griffith has assigned a provisional patent to the University of California related to the described behavioral assay. Other authors report no conflicts of interest.

Figures

Fig. 1
Fig. 1
Thermal Escape Box Apparatus. (A) An arial view of the thermal escape box showing the light chamber containing an opening for insertion of temperature-controlled plates, and the entry way into the dark chamber. (B-C) Arial views of the thermal Escape Box apparatus, and (D) a side view of the apparatus mounted on two temperature-controlled plates, with components labeled 1: Open-air light chamber, 2: Acrylic platform, 3: Temperature controlled plates, 4: Entry into dark chamber, 5: Closed dark chamber.
Fig. 2
Fig. 2
The Thermal Escape Box can be used to measure physiological thermosensing. 8–10 week old C57Bl/6 male (A, n = 21 mice; 5 °C vs. 30 °C, p = 0.0305, and 30 °C vs 52 °C, p = 0.0016) and female (B, n = 25 mice; 5 °C vs. 30 °C, p = 0.0158; 18 °C vs. 30 °C, p = 0.004; and 30 °C vs. 52 °C, p = 0.0004) mice were assayed in the thermal escape box assay at 4 different temperatures. The temperature order was as follows: 30 °C, 5 °C, 18 °C, and 52 °C. Significance was determined using a Friedman’s One-way ANOVA (A-P = 0.0030; B-P = 0.0010) with Dunn’s multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Individual dots represent biological replicates. Video 1. A C57Bl/6 mouse readily transverses plates set to a preferred temperature of 30 °C, but takes considerably longer to transverse plates set to 5 °C.
Fig. 3
Fig. 3
Starting the Thermal Escape Box Assay with a noxious temperature increases escape latencies at innocuous temperatures. 8–10-week-old C57Bl/6 mice of both sexes were assayed in the thermal escape box at 4 different temperatures. The temperature order was as follows: 5 °C, 18 °C, 30 °C and 52 °C. Significant differences were observed between 30 °C and 52 °C in males (A, n = 17 mice, p = 0.0171) and females (B, n = 20 mice; p = 0.0044). Significance was determined using a Friedman’s One-way ANOVA (A-P = 0.0164; B-P = 0.0363) with Dunn’s multiple comparisons. Individual dots represent biological replicates.
Fig. 4
Fig. 4
Habituation to the thermal escape box apparatus impairs performance. Mice were habituated to the apparatus for 30 min one day prior to behavioral testing. No significant differences in escape latencies were observed between temperatures. Male and female mice were combined (n = 9). Significance was determined using a Friedman’s One-way ANOVA (P = 0.0364) with Dunn’s multiple comparisons. Individual dots represent biological replicates.
Fig. 5
Fig. 5
Same day repeat testing in the thermal escape box lengthens escape latencies. 8–10-week-old C57Bl/6 mice of both sexes were assayed in the thermal escape box assay at two different temperatures three times in the same day, with 90 min between trials. The temperature order was as follows: 30 °C, 5 °C. (A) Significant differences were observed between the trials at 5 °C (n = 10 mice, Trial 1 vs. Trial 2, p = 0.0171; Trial 1 vs. Trial 3, p = 0.0380) and at 30 °C (n = 10 mice, Trial 1 vs. Trial 3, p = 0.0013; Trial 2 vs. Trial 3, p = 0.03469). (B) Significant differences were observed between the temperatures in the first trial (n = 10 mice, 5 °C vs. 30 °C, p = 0.0442) and the second trial (n = 10 mice, 5 °C vs. 30 °C, p = 0.0005). No significant differences in escape latencies were observed in the third trial. Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 27) = 2.774, P = 0.0802; Row Factor: F (1, 27) = 15.09, P = 0.0006; Column Factor: F (2, 27) = 7.724, P = 0.0022) with Tukey’s multiple comparisons. Individual dots represent biological replicates.
Fig. 6
Fig. 6
Repeat testing with the thermal escape box within the same week can impact performance. 8–10-week-old C57Bl/6 mice of both sexes were assayed in the thermal escape box assay at four different temperatures three times in the same week, with subsequent runs on every other day. The temperature order was as follows: 30 °C, 5 °C, 18 °C, and 52 °C. Significant differences were observed at 5 °C between Trial 1 and Trial 2 (A, n = 27 mice, p = 0.0016) and Trial 1 and Trial 3 (A, n = 27 mice, p = 0.0003), F-value = 14.15. Significant differences were observed at 18 °C between Trial 1 and Trial 3 (B, n = 27 mice, p = 0.0062). No significant differences in escape latencies were observed at 30 °C and 52 °C (C, D). Significance was determined using a Friedman’s One-way ANOVA (A-P = 0.0001; B-P = 0.0001; C-P = 0.3914; D-P = 0.3311) with Dunn’s multiple comparisons. Individual dots represent biological replicates. The average escape latencies for each temperature were: Trial 1––5 °C: 83.64 s ± 14.56 s, 18 °C: 97.14 s ± 13.2 s; 30 °C: 77.74 s ± 10.70 s, 52 °C: 138.1 s ± 11.05 s; Trial 2––5 °C: 142.8 s ± 10.64 s, 18 °C: 126.3 s ± 12.37 s; 30 °C: 77.37 s ± 13.12 s, 52 °C: 145.9 s ± 11.63 s; Trial 3––5 °C: 147.4 s ± 10.55 s, 18 °C: 133.7 s ± 11.62 s; 30 °C: 82.75 s ± 13.12 s, 52 °C: 133.4 s ± 12.18 s.
Fig. 7
Fig. 7
Validation of the thermal escape box using mice with established thermosensory deficits. 8–10-week-old TRPM8−/− and TRPV1−/− mice of both sexes were assayed in the thermal escape box assay. The temperature order for TRPM8−/− mice was as follows: 30 °C, 5 °C, 18 °C, and 52 °C. The temperature order for TRPV1−/− mice was as follows: 30 °C, 5 °C, and 52 °C. No significant differences in escape latencies were observed in TRPM8−/− males (A, n = 13 mice). Significant differences were observed in TRPM8−/− females (B, n = 14 mice, 18 °C vs. 30 °C, p = 0.0030). No significant differences in escape latencies were observed in Trpv1−/− males (C, n = 12 mice). Significant differences were observed in TRPV1−/− females (D, n = 22 mice, 5 °C vs. 30 °C, p = 0.0019). Significance was determined using a Friedman’s One-way ANOVA (A-P = 0.0746; B-P = 0.0073; C-P = 0.5097; D-P = 0.0017) with Dunn’s multiple comparisons. Individual dots represent biological replicates.
Fig. 8
Fig. 8
The thermal escape box detects the effect of thermal pain on effort-based decision making in the chronic constriction injury model. 10–12-week-old C57Bl/6 mice of both sexes were assayed in the thermal escape box assay at 12 days post CCI procedures. The temperature order was as follows: 30 °C, 18 °C, and 10 °C. Significant differences in escape latencies were observed between CCI and sham groups at 10 °C (CCI n = 30 mice and sham n = 8 mice, p = 0.0004). Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 72) = 4.817, P = 0.0109; Row Factor: F (1.119, 40.27) = 9.123, P = 0.0033; Column Factor: F (1, 36) = 4.943, P = 0.0326) with Tukey’s multiple comparisons. Filled bars represent CCI-treated mice and clear bars represent sham controls. Individual dots represent biological replicates.
Fig. 9
Fig. 9
The thermal escape box detects the effect of thermal pain on effort-based decision making in chemotherapeutic induced cold allodynia. 10–12-week-old C57Bl/6 female mice were assayed in the thermal escape box assay following induced cold allodynia from chronic oxaliplatin injection. Mice were assayed at 5 days post final injection. The temperature order was as follows: 30 °C, 18 °C, and 5 °C. Significant differences in escape latencies were observed between oxaliplatin and vehicle groups at 5 °C (oxaliplatin n = 30 mice and vehicle n = 10 mice, p = 0.02). Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 76) = 1.479, P = 0.2343; Row Factor: F (1.085, 42.23) = 11.53, P = 0.0012; Column Factor: F (1, 38) = 3.008, P = 0.0910) with Tukey’s multiple comparisons. Filled bars represent oxaliplatin-treated mice and clear bars represent vehicle controls. Individual dots represent biological replicates.
Fig. 10
Fig. 10
The thermal escape box detects the effect of thermal pain on effort-based decision making in chemotherapeutic induced cold allodynia. 10–12-week-old C57Bl/6 female mice were assayed in the thermal escape box assay at following induced cold allodynia from chronic oxaliplatin injection. Mice were assayed at 5 days post final injection. 4 h prior to being assayed, mice were injected with the analgesic meloxicam (5 mg/kg), or a saline vehicle control. The temperature order was as follows: 30 °C, 18 °C, and 5 °C. Significant differences in escape latencies were observed between meloxicam and vehicle groups at 5 °C (meloxicam n = 10 mice and vehicle n = 10 mice, p = 0.0193). Significance was determined using a Two-way ANOVA (Row Factor x Column Factor: F (2, 36) = 2.280, P = 0.1169; Row Factor: F (2, 36) = 11.53, P < 0.0001; Column Factor: F (1, 18) = 1.455, P = 0.2434) with Tukey’s multiple comparisons. Filled bars represent meloxicam-treated mice and clear bars represent vehicle controls. Individual dots represent biological replicates.

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