A novel behavioral assay for measuring cold sensation in mice

Abstract

Behavioral models of cold responses are important tools for exploring the molecular mechanisms of cold sensation. To complement the currently cold behavioral assays and allow further studies of these mechanisms, we have developed a new technique to measure the cold response threshold, the cold plantar assay. In this assay, animals are acclimated on a glass plate and a cold stimulus is applied to the hindpaw through the glass using a pellet of compressed dry ice. The latency to withdrawal from the cooled glass is used as a measure of the cold response threshold of the rodents, and the dry ice pellet provides a ramping cold stimulus on the glass that allows the correlation of withdrawal latency values to rough estimates of the cold response threshold temperature. The assay is highly sensitive to manipulations including morphine-induced analgesia, Complete Freund's Adjuvant-induced inflammatory allodynia, and Spinal Nerve Ligation-induced neuropathic allodynia.

Figure 1. The Cold Plantar Assay.

A. Grind dry ice into a fine powder with a hammer (panel 1). Load the dry ice powder into a 3mL syringe and compress against a flat surface until powder cannot be compressed any further (panel 2). Using the syringe plunger, push the dry ice pellet 20–30mm past the tip of the syringe (panel 2). Gently but firmly apply the flat end of the dry ice pellet to the glass surface underneath the paw of the mouse (panel 3) and measure the latency to withdrawal with a stopwatch. B. Schematic of the cold plantar assay apparatus. Mice are acclimated in plastic enclosures on a glass plate. A mirror is placed underneath the apparatus to facilitate targeting of the dry ice probe.

Figure 2. The Cold plantar assay applies a consistent ramping cold stimulus.

A. The cold plantar withdrawal latency increases as thicker glass plates are used. With Swiss Webster mice, on the 1/8″ glass the average latency is 3.79±0.5 seconds, on the 3/16″ glass the average latency is 7.29±0.9 seconds, and on the 1/4″ glass the average latency is 11.63±1.3 seconds. The average latencies on each glass are significantly different (1-way ANOVA with Bonferroni post-hoc test ***p<.0001 between all thicknesses; n = 12 mice per glass). B. With C57 mice, on the 1/8″ glass the average latency is 3.83±0.5 seconds, on the 3/16″ glass the average latency is 7.0±1.4 seconds, and on the 1/4″ glass the average latency is 10.4±0.9 seconds. The average latencies on each glass are significantly different (1-way ANOVA with Bonferroni post-hoc test ***p<.0001 between all thicknesses; n = 10 mice per glass). C. Schematic showing how the temperature between the paw and the glass was measured. D. Curves representing the change in temperature between the paw and the glass during and shortly after dry ice stimulation. For each curve, the dry ice stimulus begins at x = 1, and ends at the colored arrow for that thickness (from 2A, 1/8″: 3.79 seconds, 3/16″: 7.29 seconds, 1/4″: 11.63 seconds). Based on these curves, mice withdraw after a 1.3°C (1/8″), 1.5°C (3/16″), or 2°C (1/4″) change in temperature (n = 6 per glass thickness).

Figure 3. The cold plantar assay requires direct paw-glass contact.

A. Schematic diagramming the experimental design. B–D. The temperature during cold plantar stimulus underneath the paw was measured on all three glass thicknesses under normal conditions, and with styrofoam spacers propping the paw away from the glass surface. In all cases, propping the paw away from the glass caused a dramatic decrease in the cold stimulus measured at the paw (n = 6 per glass thickness). E. The temperature underneath the paw was measured during Hargreaves radiant heat stimulation with the paw propped up with styrofoam. Unlike the cold plantar assay, the thermal stimulus in the Hargreaves assay is largely unaltered when the paw is propped away from the glass (n = 6).

Figure 4. The cold plantar assay can measure analgesia and allodynia.

A. Mice were given subcutaneous injections of 1.5mg/kg morphine or saline. Thirty minutes after injection, morphine-injected mice had significantly higher withdrawal latencies on the 1/4″ glass, but 60 minutes after injection the morphine injected mice had returned to baseline latency values (2-way ANOVA main effect *p<0.05 with Bonferroni post-hoc test; 30 minutes **p<0.01; n = 12 per group). B. Mice were given intraplantar injections of Complete Freund's Adjuvant or saline. 2 and 3 hours after injection, CFA-injected mice had significantly lower withdrawal latencies on the 1/4″ glass than the saline-injected controls (2-way ANOVA main effect p<.0001 with Bonferroni post-hoc test; 120 minutes *p<0.05, 180 minutes **p<0.01 n = 12 per group). 4 hours after intraplantar injection, all animals were given subcutaneous injections of 1.5mg/kg morphine. At 4.5 hours both CFA- and Saline-injected animals had significantly increased withdrawal latencies compared to their values at 3 hours (1-way ANOVA with Dunnett's post-hoc test; CFA 3hours vs. CFA 4.5 hours $$$ p<0.0001, Saline 3 hours vs. Saline 4.5 hours $$$ p<0.0001). At 5.5 hours, the CFA-injected mice had significantly lower withdrawal latencies than their saline-injected counterparts (2-way ANOVA with Bonferroni post-hoc test; 5.5 hours **p<0.01). 24 hours after intraplantar injection, the CFA-injected mice still had significantly lower withdrawal latencies than their saline-injected counterparts, but this difference resolved by 48 hours (2-way ANOVA with Bonferroni post-hoc test; 24 hours ** p<0.01, 48 hours p>0.05).

Figure 5. The cold plantar assay can measure L4 spinal nerve ligation induced allodynia.

Mice underwent the spinal nerve ligation (SNL) procedure or a sham procedure on day 0. A. On days 3, 6, and 10 post surgery SNL mice had significantly lower cold plantar withdrawal latencies on the 1/4″ glass than sham mice (2-way ANOVA main effect ***p<0.001 with Bonferroni post-hoc test; 3d ***p<0.001, 6d **p<0.01, 10d **p<0.01; Sham n = 5, SNL n = 6). After baseline measurements on day 10 post surgery, mice were injected with 1.5mg/kg morphine. Thirty minutes after morphine injection, both SNL and sham mice had withdrawal latencies that were significantly elevated from their baseline values that day (1-way ANOVA with Dunnett's post-hoc test; Sham $ p<0.05, SNL $$ p<0.01). By 90 minutes after morphine injection, the SNL mice again had significantly decreased withdrawal latencies than the sham mice (2-way ANOVA with Bonferroni post-hoc test; ***p<0.001). B. On days 2, 5, and 11 post surgery SNL mice spent more time flicking after acetone application than sham mice (2-way ANOVA main effect *p<0.05 with Bonferroni post-hoc test; 2 days p>0.05, 5 days p>0.05, 11 days p>0.05). After baseline measurements on day 11 post surgery, mice were injected with 1.5mg/kg morphine. There was a significant reduction in the time responding to acetone in the SNL group but not in the Sham group (1-way ANOVA with Dunnett's post-hoc test; Sham p>0.05, SNL $$ p<0.01).