Mouse mast cells and mast cell proteases do not play a significant role in acute tissue injury pain induced by formalin

Abstract

Subcutaneous formalin injections are used as a model for tissue injury-induced pain where formalin induces pain and inflammation indirectly by crosslinking proteins and directly through activation of the transient receptor potential A1 receptor on primary afferents. Activation of primary afferents leads to both central and peripheral release of neurotransmitters. Mast cells are found in close proximity to peripheral sensory nerve endings and express receptors for neurotransmitters released by the primary afferents, contributing to the neuro/immune interface. Mast cell proteases are found in large quantities within mast cell granules and are released continuously in small amounts and upon mast cell activation. They have a wide repertoire of proposed substrates, including Substance P and calcitonin gene-related peptide, but knowledge of their in vivo function is limited. We evaluated the role of mouse mast cell proteases (mMCPs) in tissue injury pain responses induced by formalin, using transgenic mice lacking either mMCP4, mMCP6, or carboxypeptidase A3 (CPA3), or mast cells in their entirety. Further, we investigated the role of mast cells in heat hypersensitivity following a nerve growth factor injection. No statistical difference was observed between the respective mast cell protease knockout lines and wild-type controls in the formalin test. Mast cell deficiency did not have an effect on formalin-induced nociceptive responses nor nerve growth factor-induced heat hypersensitivity. Our data thus show that mMCP4, mMCP6, and CPA3 as well as mast cells as a whole, do not play a significant role in the pain responses associated with acute tissue injury and inflammation in the formalin test. Our data also indicate that mast cells are not essential to heat hypersensitivity induced by nerve growth factor.

Keywords: Pain formalin; mast cell; protease; transgenic.

Figure 1.

mMCP4^−/−, mMCP6^−/−, and CPA3^−/− mice do not differ from controls in the formalin test. Twenty microliters of 5% formalin were injected subcutaneously into the right hind paw and the mouse was then observed for pain behavior for 60 min. (a) mMCP4^−/− mice (n = 8) show similar pain behavior to controls (n = 9) over the time course of the formalin test, except with a slight trend for higher pain responses in the last 15 min of the test. (b) In contrast to the mMCP4^−/− mice, the mMCP6^−/− mice (n = 9) had a slight tendency to show lower pain responses than controls during the inflammatory phase, but the difference was not significant (p > 0.05). (c) The pain behavior of CPA3^−/− mice (n = 6) was not significantly different from controls. (d) and (e) No differences were observed between genotypes in the total pain behavior in Phase 1 (0–10 min) or in the inflammatory Phase 2 (10–60 min). The same set of control mice (n = 9) was used in all comparisons and presented in (a), (b), and (c) to facilitate visual presentation. Kruskal–Wallis and Dunn’s post hoc tests were used. Data are presented as mean ± SEM. WT: wild type.

Figure 2.

Mast cell-deficient mice do not differ from controls in the formalin test or in heat hypersensitivity induced by NGF. (a) Toluidine blue-stained cryo sections of hind paws. Plantar surfaces of hind paws of naïve Mcpt5^Cre-;R-DTA (controls, n = 2) and Mcpt5^Cre+;R-DTA (n = 2) were fixed with PFA, embedded in OCT compound, cut into 12-µm-thick sections, and subsequently stained with toluidine blue. Red arrows point to metachromatically stained mast cells in control tissue, which are absent in tissue of Mcpt5^Cre+;R-DTA mice. 10× objective, scale bar = 50 µm. Inset represents metachromatically stained mast cell, 20× objective, scale bar = 20 µm. (b) The pain behavior of mast cell-deficient mice (Mcpt5^Cre+;R-DTA, n = 8) does not differ from controls (n = 10) over the time course of the formalin test.(Continued)Slightly lower responses were observed at 30–35 min, but the difference was not significant (Mann–Whitney: p = 0.10). (c) and (d) When total pain behavior in Phase 1 (0–10 min) or in the inflammatory Phase 2 (10–60 min) was analyzed, no differences were observed between genotypes. (e–f) Hargreaves test coupled with NGF injection on Mcpt5^Cre+;R-DTA mice (n = 7), and controls (n = 6). (e) The baseline Hargreaves values before NGF injection (“B” on the x-axis) did not differ between the two genotypes (Student’s t test: p = 0.64). After NGF injection, only Mcpt5^Cre+;R-DTA mice developed heat hypersensitivity compared with their baseline values, at 4-h post-injection (one-way repeated measurement ANOVA: p < 0.05). (f) Despite the absence of significant hypersensitivity in the controls, there was no difference observed between the two genotypes at any time point before or after the injection (Student’s t test: p > 0.05). All data are presented as mean ± SEM. NGF: nerve growth factor.