Article Figures & Data
Figures
- Figure 1.
Assessment of the effect of pregabalin on acute oxaliplatin-induced cold allodynia. A, Cold-plate assessment at 10°C of mice treated with oxaliplatin and successively either vehicle or 2 mg/kg of pregabalin. Activity was measured as the total number of nociceptive behaviors (hindpaw lifts, shakes, licks) over the test duration. A cutoff time of 300 s was used to limit tissue damage. The baseline data in the graph refers to the mice injected with oxaliplatin. For the baseline versus oxaliplatin-treated animal data please refer to Extended Data Figure 1-1. B, Plot showing the decrease of the number of nociceptive behaviors over time following vehicle or pregabalin injection. C, Cold-plate assessment at 10°C of mice treated only with pregabalin. Activity was measured as the total number of forepaw lifts over the test duration. A cutoff time of 300 s was used to limit tissue damage. n = 8 oxaliplatin + pregabalin mice, n = 8 oxaliplatin + vehicle mice, n = 6 pregabalin only mice. Statistical analyses in A and B were performed using one-way and two-way ANOVA tests with multiple comparisons, respectively. Statistical Analysis in C was performed using paired t test. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 2.
Pregabalin treatment decreases both the number of neurons responding to cold stimuli and the intensity of their responses. A, Example images showing the reduction in the population of DRG neurons responding to ice-water 50 min after treatment with 2 mg/kg of pregabalin. B, Graph showing the decrease of the percentages of cell responding to ice-water stimulus. C, Example traces showing the decrease in the intensity of the calcium signals over time from pregabalin injection. D, Plot showing the mean response amplitude before (black trace) and 40 min (blue) and 50 min (pink) after pregabalin injection. n = 45 cells for all time points. E, Graphs showing the ΔFmax changes before and 40 and 50 min after pregabalin injection. F, Graphs showing the area under curve changes before and 40 and 50 min after pregabalin injection. n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice. Statistical analysis in B was performed using repeated measures ANOVA test with multiple comparisons. Statistical analysis in D was performed using multiple Wilcoxon tests. Statistical analyses in E and F were performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For the data regarding acetone, hot water, and mechanical stimulations please refer to Extended Data Figures 2-1 and 2-2.
- Figure 3.
Pregabalin treatment significantly reduces the excitability of cold responding cells and targets preferentially medium-large mechano-cold sensors. A, Example traces of a representative cold-sensing cell in response to a series of 4°C temperature drops from a holding temperature of 32°C. The traces represent the change in the threshold of this neuron over time (0–50 min) from pregabalin injection. Arrowheads mark the first response to temperature drops in every timepoint. B, Graph showing the change of the relationship between the number of cold-responding neurons and the temperature drop over time from pregabalin injection. While the relationship at baseline can be fit with a linear equation (y = −3.292× + 99.84; r2 = 0.9880; n = 115), the one at 50-min fits a quadratic equation (y = 110.8 − 7.381× + 0.1265x2; r2 = 0.9927; n = 109). The slopes at baseline and 50 min after treatment are significantly different (***p < 0.001), as analyzed by Wilcoxon test. For a complete visualization of the data regarding the cold threshold changes in all time points please refer to Extended Data Figure 3-1. C, Graphs showing the change in the number of total mechano-responding neurons that respond also to ice-water stimulus. Mice treated with vehicle do not exhibit the decrease over time from treatment as the mice treated with pregabalin do. For the graphs showing the mechano-acetone and mechano-heat responding cells trends please refer to Extended Data Figure 3-2. D, Numeric plots of the distribution of cold-responding cell areas in oxaliplatin-treated mice before (black trace) and after 50 min (pink trace) from pregabalin injection. The difference between the distributions at different timepoints after treatment is statistically significant (***p = 0.0001), as analyzed by Wilcoxon test. For a complete visualization of the numeric distributions of cold responding cells areas in all time points please refer to Extended Data Figure 3-3. n = 7 mice for temperature threshold experiments; n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice.
- Figure 4.
Knock-out of the α2δ1 subunit of the VGCCs is sufficient to abolish the effects of pregabalin both on cold allodynia and on silencing of cold responding DRG neurons. A, Plot showing the decrease of the number of nociceptive behaviors over time from vehicle (n = 8; black trace) or pregabalin injection in WT [n = 8 (blue trace) + 10 (light blue trace)] and α2δ1-KO mice (n = 10; red trace) when exposed to a 10°C cold plate for 5 min (**p < 0.01). B, Example images showing that the reduction of the population of DRG neurons responding to ice-water 50 min after treatment with pregabalin is absent in α2δ1-KO mice. C, Graph showing the disappearance of the decrease of the percentages of cell responding to ice-water stimulus in α2δ1-KO mice. Change is quantified as fold-change to baseline. D, Numeric plots of the distribution of cold responding cell areas in oxaliplatin-treated α2δ1-KO mice before (gray trace) and 50 min after pregabalin injection (light pink trace). n = 7 α2δ1-KO mice, n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice for imaging data. Statistical analyses in C were performed using repeated measures ANOVA test with multiple comparisons. *p < 0.05 (WT vehicle vs WT pregabalin treated); ##p < 0.01 (α2δ1-KO vs WT pregabalin treated). For the data regarding acetone, hot water, and mechanical responses, please refer to Extended Data Figure 4-1. For the data regarding the area distribution of cold responding cells after pregabalin treatment in α2δ1-KO animals, please refer to Extended Data Figure 4-2.
Extended Data
Extended Data Figure 1-1
Assessment of cold allodynia after injection of oxaliplatin. Cold-plate assessment at 10°C of mice treated with oxaliplatin. Activity was measured as the total number of nociceptive behaviors (shaking, lifting, licking, guarding, biting) over the test duration. A cutoff time of 300 s was used to limit tissue damage. n = 10 oxaliplatin-treated mice. Statistical analysis was performed using paired t test. ****p < 0.0001. Download Figure 1-1, TIF file.
Extended Data Figure 2-1
Pregabalin treatment decreases the number of neurons responding to a chemical cold stimulus. A, Example images showing the reduction in the population of DRG neurons responding to acetone 50 min after treatment with 2 mg/kg of pregabalin. B, Graph showing the decrease of the percentages of cell responding to acetone. n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice. Statistical analysis in B was performed using repeated measures ANOVA test with multiple comparisons. *p < 0.05, **p < 0.01. Download Figure 2-1, TIF file.
Extended Data Figure 2-2
Pregabalin treatment does not affect other sensory modalities besides cold. A, Example images showing no change in the population of DRG neurons responding to mechanical pinch 50 min after treatment with 2 mg/kg of pregabalin. B, Graph showing the unchanged percentages of cell responding to mechanical pinch. C, Example images showing no change in the population of DRG neurons responding to a 55°C water stimulus 50 min after treatment with 2 mg/kg of pregabalin. D, Graph showing the unchanged percentages of cell responding to a 55°C water stimulus. n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice. Statistical analysis in B and D was performed using repeated measures ANOVA test with multiple comparisons. Download Figure 2-2, TIF file.
Extended Data Figure 3-1
Pregabalin treatment increases temperature threshold of cold-responding cells over time. Graph showing the change of the relationship between the number of cold-responding neurons and the temperature drop over time from pregabalin injection. Download Figure 3-1, TIF file.
Extended Data Figure 3-2
Pregabalin treatment reduces the number of polymodal mechano-cold but not mechano-heat responding cells. A, Graph showing the change in the number of total mechano-responding neurons that respond also to acetone stimulus. Mice treated with vehicle do not exhibit the decrease over time from treatment as the mice treated with pregabalin do. B, Graph showing the change in the number of total mechano-responding neurons that respond also to a 55°C water stimulus. Mice do not exhibit significant differences in the percentage of polymodal mechano-heat responding neurons over time from pregabalin treatment. n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice. Download Figure 3-2, TIF file.
Extended Data Figure 3-3
Pregabalin preferentially inhibits the activity of “silent” cold-sensing neurons. A, Cumulative plots of the cell areas in oxaliplatin-treated mice before and after pregabalin injection. The difference between the distributions at different timepoints after treatment is statistically significant (p < 0.0001), as analyzed by Kruskal–Wallis test. B, Number plots of the distribution of cell areas of cold-responding cells in oxaliplatin-treated mice before and after pregabalin injection. The difference between the numeric distributions at different timepoints after treatment is statistically significant (p < 0.0001), as analyzed by Kruskal–Wallis test. C, Graph showing the area under curve changes before and 50 min after pregabalin injection. The cell areas are divided into basal cold responding cells (A < 480 μm2) and silent cold sensors (A > 480 μm2). Albeit there is a difference in both populations after 50 min from treatment, the population of silent cold sensors seems to be silenced almost completely. These thresholds have been calculated previously (MacDonald et al., 2021). Download Figure 3-3, TIF file.
Extended Data Figure 4-1
Knock-out of the α2δ1 subunit of the VGCCs abolishes the effects of pregabalin on cold but has no effect on heat and mechanical responses. A, Example images showing no change in the population of DRG neurons responding to acetone, 55°C water, and mechanical pinch 50 min after treatment of α2δ1-KO mice with 2 mg/kg of pregabalin. B, Graph showing the unchanged percentages of cell responding to acetone in α2δ1-KO mice with respect to WT ones. C, Graph showing the unchanged percentages of cell responding to a 55°C water stimulus in α2δ1-KO mice with respect to WT ones. D, Graph showing the unchanged percentages of cell responding to mechanical pinch in α2δ1-KO mice with respect to WT ones. n = 7 α2δ1-KO mice, n = 6 pregabalin-treated mice, n = 5 vehicle-treated mice for imaging data. Statistical analyses in B–D were performed using repeated measures ANOVA test with multiple comparisons. ##p < 0.01 (α2δ1-KO vs WT pregabalin treated). Download Figure 4-1, TIF file.
Extended Data Figure 4-2
Knock-out of the α2δ1 subunit of the VGCCs abolishes the silencing effect of pregabalin on silent cold sensors. Graph showing the area under curve changes before and 50 min after pregabalin injection in the α2δ1-KO mice. The cell areas are divided into basal cold responding cells (A < 480 μm2) and silent cold sensors (A > 480 μm2). There seems to be no difference in the population of basal and silent cold sensors after pregabalin treatment when the α2δ1 subunit is knocked down globally. These thresholds have been calculated previously (MacDonald et al., 2021). Download Figure 4-2, TIF file.
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