A novel operant-based behavioral assay of mechanical allodynia in the orofacial region of rats

Abstract

Background: Detecting behaviors related to orofacial pain in rodent models often relies on subjective investigator grades or methods that place the animal in a stressful environment. In this study, an operant-based behavioral assay is presented for the assessment of orofacial tactile sensitivity in the rat.

New methods: In the testing chamber, rats are provided access to a sweetened condensed milk bottle; however, a 360° array of stainless steel wire loops impedes access. To receive the reward, an animal must engage the wires across the orofacial region. Contact with the bottle triggers a motor, requiring the animal to accept increasing pressure on the face during the test. To evaluate this approach, tolerated bottle distance was measured for 10 hairless Sprague Dawley rats at baseline and 30 min after application of capsaicin cream (0.1%) to the face. The experiment was repeated to evaluate the ability of morphine to reverse this effect.

Results: The application of capsaicin cream reduced tolerated bottle distance measures relative to baseline (p<0.05). As long as morphine did not cause reduced participation due to sedation, subcutaneous morphine dosing reduced the effects of capsaicin (p<0.001). Comparison with existing method: For behavioral tests, experimenters often make subjective decisions of an animal's response. Operant methods can reduce these effects by measuring an animal's selection in a reward-conflict decision. Herein, a method to measure orofacial sensitivity is presented using an operant system.

Conclusions: This operant device allows for consistent measurement of heightened tactile sensitivity in the orofacial regions of the rat.

Keywords: Allodynia; Behavior; Operant; Orofacial; Pain.

Figure 1. An operant device for the detection of orofacial sensitivity to mechanical stimuli

To engage with a reward bottle filled with sweetened condensed milk, an animal must engage with a 360° degree array of 0.010″ diameter looped stainless steel wires. This design minimizes error due to animal avoidance behavior (A). When the animal engages the array (B), a motor and threaded rod moves the reward bottle assembly farther from the cage (C). The animal must continue to move its head through mechanical stimulus wires to receive the reward bottle.

Figure 2. Reward bottle tolerance distance with and without the application of capsaicin cream to the orofacial regions of a Sprague-Dawley rat

Following the application of capsaicin cream to the orofacial region of a rat’s face, reward bottle tolerance distances were reduced (Panel A, p-value associated with capsaicin treatment from 2-way Main-Effects ANOVA). Panel B shows that each application of capsaicin resulted in reduced tolerance distance relative to Baseline Day 1 (indicated by a “1”), Baseline Day 3 (indicated by a “3”), and Baseline Day 4 (indicated by a “4”); only the first application of capsaicin was different from Baseline Day 2 (indicated by a “2”, Tukey’s HSD post-hoc test). Data presented in Panel C demonstrate changes in individual animal tolerance distance resulting from capsaicin. Capsaicin application had a significant effect on tolerance distance in 7 of 10 animals (p<0.05, paired t-test) and was near significant in an additional 2 animals (0.05

Figure 3. Morphine attenuation of capsaicin-induced orofacial hypersensitivity

Average tolerated bottle distances are shown in Figure 3, Panel A. At high morphine doses, animals reduced their participation in the test (see x’s and dotted line). At 5 mg/kg, 3 of 4 animals fully participated, while 2 of 4 animals fully participated at 15 mg/kg and 1 of 4 animals fully participated at 20 mg/kg. No animal fully participated when receiving 25 mg/kg. In Panel B, trials where animals failed to fully participate are removed. With this exclusion, treatment with morphine demonstrated an ability to reduce the effects of capsaicin application to the orofacial regions (p<0.001, main effect in ANOVA, Panel C). While all morphine doses visually appear to reduce the shift in tolerated bottle distance to near baseline levels (grey squares are closer to zero than the white square), only morphine doses of 10 mg/kg resulted significant changes in bottle distance relative to no morphine treatment controls (*, p<0.001, Panel B).