Focal Inflammation Causes Carbenoxolone-Sensitive Tactile Hypersensitivity in Mice

Abstract

A focal and transitory inflammation induced by injection of complete Freund's adjuvant (CFA) in the submandibular skin of mice elicits pain behavior that persists for several weeks after the initial inflammation has resolved. Chronic pain, assessed as tactile hypersensitivity to stimulation with von Frey filaments, was evident from 1-7 weeks following CFA injection, although inflammation at the injection site was resolved by 3-4 weeks. In contrast, there were no changes in tactile sensitivity in the paw (un-injected site for comparison), no alterations in open field behavior and no differences in a functional observation battery evident in CFA-treated mice compared to controls (saline-injected) or to baseline (before CFA injection). Neither strain (Balb/c vs. C57BL/6) nor sex differences in baseline tactile threshold were significant in the submandibular skin. CFA-induced tactile hypersensitivity was also not a function of strain or sex. A single intraperitoneal injection of the gap junction blocker carbenoxolone (CBX) restored normal tactile thresholds in CFA-treated mice when administered at the peak of inflammation (1 week), after significant resolution of inflammation (3 weeks) or after total resolution of inflammation (4 and 5 weeks) without altering the tactile threshold of control subjects, tactile threshold in the paw or open field behavior. Thus, in this novel model of post-inflammatory pain, transitory inflammation induced persistent sex- and strain-independent behavioral hypersensitivity that was reversed by the gap junction blocker CBX, suggesting neuronal and/or glial plasticity as a major component of the chronic pain.

Fig. (1)

The time course of inflammation assessed as density of white blood cells in H&E-stained sections before (Pre) and for 5 weeks (wk) after CFA injection (A). In Balb/c mice, inflammation peaked at 1 week after CFA injection, was significantly reduced 2–3 weeks after CFA injection, and was completely resolved 4–5 weeks after CFA injection when compared to either Pre injection values or compared to saline (Sal) injected controls at the same time points. (B) In C57BL/6 mice inflammation also peaked at 1 week, but was significantly resolved by 3 weeks. * indicates significant differences from Pre. ^ indicates significant differences from CFA 1 week. ⁺ indicates significant differences from saline at a given time point. Panels C–F illustrate representative staining from non-injected Balb/c mice (C), staining 1 week post-Sal injection (D), 1 week post-CFA injection (E) and 4 weeks post-CFA injection in Balb/c mice (F). Insert in E shows polymorphonuclear neutrophils (*), macrophages (arrowheads) and lymphocytes (arrow) infiltrating the submandibular skin. Scale bar=100 µm

Fig. (2)

Influence of strain and sex on baseline tactile sensitivity in the submandibular skin assessed with von Frey filaments: n=8 in each condition for this and the following graph. Number (#) of withdrawal responses out of 10 stimulations across all stimulus intensities in Balb/c and C57BL/6 mice are shown in panels A–C. Strain did not influence the number of responses in the submandibular skin (B), but sex affected this measure significantly (C: p<0.04). Tactile thresholds for submandibular skin in Balb/c and C57BL/6 mice of both sexes are shown in panels D–F as the stimulus intensity resulting in 8/10 withdrawal responses. There were no significant effects of strain (E) or sex (F) on baseline tactile threshold in the submandibular skin. There were no interactions between sex and strain.

Fig. (3)

Influence of strain and sex on baseline (before CFA or saline injection) tactile sensitivity in the hind paw assessed with von Frey filaments. Number (#) of withdrawal responses is shown in panels A–C. Strain (B) significantly influenced the number of responses evoked in the paw (p<0.005), but there was no significant effect of sex (C). Tactile threshold for paw is shown in panels D–F. There was also a significant effect of strain on the paw tactile threshold (E, p<0.03), but no main effect of sex (F). There were no interactions between sex and strain.

Fig. (4)

Tactile thresholds in the submandibular skin (A) and paw (B) after CFA or saline injection into the submandibular skin over 7 weeks. Data from both strains are combined. CFA reduced tactile thresholds in the submandibular skin for up to 7 weeks, but had no effect on paw tactile thresholds at any time point. * indicates significant differences between Pre and post CFA injections (p<0.007). ⁺ indicates significant differences between CFA- and saline-injected mice (p<0.001). Samples sizes were: n=16–28 for saline (1–5 weeks); n=68 for CFA (Pre and 1 week) and n=20–44 for CFA (2–7 weeks).

Fig. (5)

Assessment of tactile thresholds of the submandibular skin before (Pre) and 1 week after CFA injection in 3 experiments each performed by a different experimenter (A,B,C) using different cohorts demonstrated the reliability of the CFA-induced tactile sensitivity and internal validity. * indicates significant differences between tactile thresholds Pre and 1 week after CFA injection (p<0.05). Experimenter A tested male Balb/c mice, B used male and female Balb/c mice and C57BL/6 mice, C used only male C57BL/6 mice.

Fig. (6)

The gap junction blocker carbenoxolone (CBX; 100 mg/kg, i.p.) reversed CFA-induced tactile hypersensitivity. (A) CBX significantly increased tactile thresholds when injected once at 1,3,4 or 5 weeks after CFA administration (* p<0.01). Tactile sensitivity was always determined 1 h after i.p. injection of CBX or saline (control). (B) The significantly lower tactile threshold evident in CFA-treated mice (CFA/CBX−) compared to controls (Sal/CBX− and Sal/CBX+) was restored to control levels after CBX injection (CFA/CBX+; p<0.01 for all comparisons). * indicates significant differences between CFA/CBX− and CFA/CBX+. ⁺ indicates significant differences between Sal/CBX−and CFA/CBX−. ^ indicates significant differences between CFA/CBX− and Sal/CBX+. Note that CBX did not alter the threshold in control mice (Sal/CBX+ and Sal/CBX−). The paw threshold was not altered by CBX in saline or CFA treated mice 4 weeks (C) or 5 weeks (D) after CFA administration.

Fig. (7)

A single i.p. injection of CBX (100 mg/kg) did not affect activity (total track length: A), exploration (number of rears: B) or thigmotaxis (anxiety-like behavior: C) in CFA treated subjects assessed in an open field test. Behavior of mice in the FOB and open field were immediately determined after tactile sensitivity tests. n=16 per condition.

Fig. (8)

Low doses of CBX (10, 25 and 50 mg/kg) are effective at reversing CFA-induced tactile hypersensitivity. Tactile sensitivity was determined 1 h after i.p. injection of CBX or saline (control). n=3–4 per group.