Influence of social interaction on nociceptive-induced changes in locomotor activity in a mouse model of acute inflammatory pain: Use of novel thermal assays

Abstract

Most acute and chronic animal models of pain rely heavily on reflexive assays for evaluating levels of nociception, which involves removing the animal from its normal social environment. Here, we examine and characterize the influence of social interactions on inflammatory pain-evoked changes in movement in two different mouse strains. To produce inflammatory nociception, we injected CFA bilaterally into the hind paws of Balb/c and C3H mice and then recorded exploratory locomotor activity using an automated detector system to first evaluate the effects of social behavior on nociception. Secondly, we determined if carprofen administration altered the effects of social behavior on nociceptive-evoked movement. This methodology was expanded to create a novel thermal activity assay to objectively measure the effect of heat and cold on CFA-evoked animal movement in paired animals. Paired Balb/c and C3H mice exhibited significant hyper-locomotion that lasted for 3h post-injection in Balb/c, but only 1h post-injection in C3H. Single Balb/c mice only showed increased activity for 1h post-injection, while single C3H mice showed no increase. This CFA-induced increase in activity in paired animals was highly inversely correlated with mechanical allodynia as measured using standard Von Frey filaments. Carprofen administration completely blocked this CFA-induced hyperlocomotor activity. Both heat and cold induced a significant increase in locomotor activity in paired mice injected with CFA, while having no effect on activity in control mice injected with saline. The results presented here indicate that social interactions greatly influence inflammatory pain-induced changes in locomotor activity and indicate that the use of movement-based assays to evaluate nociception in paired mice may provide an alternative and more sensitive method to quantify nociception and characterize novel analgesic effects over time in the context of social interactions in rodent models of pain.

Fig. 1. Assessment of social interactions on normal locomotor behavior in Balb/c and C3H mouse strains

Exploratory activity was averaged over 4 hours in naïve Balb/c and C3H single or paired animals in an activity chamber. Paired Balb/c animals moved significantly less than strain-matched single animals (**p < 0.01). Inter-strain comparisons revealed increased activity in single Balb/c animals compared to C3H singles (**p < 0.01). Data shown as mean ± SEM; n = 6/single, n = 12/paired; student's unpaired t-test.

Fig. 2. Balb/c, but not C3H social interactions significantly influence nociceptive-induced changes in locomotor activity

To evaluate the social effects of bilateral CFA injection on locomotor activity in Balb/c and C3H animals, single or paired animals from each strain were placed into activity chambers and the numbers of locomotor movements (horizontal activity counts) were assayed over a 4-hour time period. When activity was quantified and averaged over a 4-hour time period, both Balb/c CFA-injected single mice (**p < 0.01) and paired mice (***p < 0.001) showed a significant increase in locomotor activity compared to their saline injected counterparts, but the effect was more significant in paired mice when compared to their respective counterparts and to CFA-injected single animals (*p < 0.05). These single and paired effects were not observed in C3H (p > 0.05). Data shown as mean ± SEM; n = 6/single, n = 12/paired; student's unpaired t-test.

Fig. 3. Temporal patterns of the effect of social interaction on CFA-induced locomotor activity

When temporal locomotor activity patterns were analyzed within strains in the context of single versus paired (socially interactive) animals in 1 hour bins over the 4-hour testing period, the activity of singly chambered Balb/c mice was only significantly increased for the first hour following injection (A), while paired mouse activity was significantly maintained over the first 3 hours of testing (C). Unlike Balb/c animals, single C3H animals showed no differences in movement (B), however; paired animals showed increased movement during the first hour of activity when compared to saline counterparts (D). Data shown as mean ± SEM; n = 6/single, n = 12/paired; *p < 0.05, ***p < 0.001; two-way ANOVA.

Fig 4. CFA-induced hyperalgesia inversely correlates with locomotor activity

The developmental time course of acute mechanical allodynia in Balb/c (A) and C3H (B) mice. Single and paired animals were assayed for exploratory locomotor activity at 1, 2, 3 and 4 hours post-CFA injection and movement was correlated to nociceptive mechanical allodynia scores attained within each strain indicated. Von Frey scores were highly and significantly inversely correlated with activity in paired mice indicating a clear relationship between mechanical allodynia and activity. Data shown as mean ± SEM; r² values indicated in graphs, n = 6/single, n = 12/paired, p < 0.05; student's unpaired t-test and linear regression analysis.

Fig 5. Administration of carprofen blocks the development of hyperlocomotor activity caused by acute noxious inflammation

Locomotor activity was assayed for 4 hours following either bilateral CFA injection or physiological saline in paired Balb/c animals. Carprofen (5 mg/kg, s.c.) was administered 10 min after injection of CFA or saline subcutaneous injection of carprofen significantly reduced CFA-induced hyperlocomotor activity over a 4 hour time period (A) and for a period of 3 hours post-injection when the data was analyzed in 1 hour bins (B). Data shown as mean ± SEM, n = 12/group; **p < 0.01, ***p < 0.001, ****p < 0.0001; student's unpaired t-test (A) and two-way ANOVA (B).

Fig. 6. The thermal locomotor assay (TLA) provides better sensitivity than traditional reflexive testing in detecting thermal hyperalgesia

A thermal plate device was inserted in an activity box allowing the measurement (B & C and E & F) of the effects of thermal stimulation on locomotor activity at the same temperatures used for the thermal reflexive testing (A & D). The effect of CFA-induced thermal hyperalgesia on locomotor activity was evaluated at baseline (BL) and at 2, 4 and 6 hours in single (B & E) and paired mice (C & F) following bilateral saline or CFA injection (n = 12/group). Single animals injected with CFA exposed to the 53°C hot plate displayed a significant decrease in PWL compared to the PWL of saline-injected control mice (A). Conversely CFA-injected animals did not display significant changes in the number of spontaneous behaviors when exposed to a 4°C cold plate over a 2-min testing period (D). In contrast, there was a significant increase in paired locomotor activity only in CFA-injected animals compared to saline-injected control mice at both 2 and 4 hours post-injection at both temperatures (C & F). Thus, locomotor activity provides equivalent or better sensitivity to traditional thermal reflexive tests, accounts for social interactions and is without the subjectivity that is often associated with reflexive thermal testing. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA