ASIC1 and ASIC3 play different roles in the development of Hyperalgesia after inflammatory muscle injury

Abstract

Acid-sensing ion channels (ASICs) respond to acidosis that normally occurs after inflammation. We examined the expression of ASIC1, ASIC2, and ASIC3 mRNAs in lumbar dorsal root ganglion neurons before and 24 hours after carrageenan-induced muscle inflammation. Muscle inflammation causes bilateral increases of ASIC2 and ASIC3 but not ASIC1 (neither ASIC1a nor ASIC1b) mRNA, suggesting differential regulation of ASIC1 versus ASIC2 and ASIC3 mRNA. Similar mRNA increases were observed after inflammation in knockout mice: ASIC2 mRNA increases in ASIC3-/- mice; ASIC2 and ASIC3 mRNAs increase in ASIC1-/- mice. Prior behavioral studies in ASIC3-/- mice showed deficits in secondary hyperalgesia (increased response to noxious stimuli outside the site of injury) but not primary hyperalgesia (increased response to noxious stimuli at the site of injury). In this study, we show that ASIC1-/- mice do not develop primary muscle hyperalgesia but develop secondary paw hyperalgesia. In contrast, and as expected, ASIC3-/- mice develop primary muscle hyperalgesia but do not develop secondary paw hyperalgesia. The pharmacological utility of the nonselective ASIC inhibitor A-317567, given locally, was tested. A-317567 reverses both the primary and the secondary hyperalgesia induced by carrageenan muscle inflammation. Thus, peripherally located ASIC1 and ASIC3 play different roles in the development of hyperalgesia after muscle inflammation.

Perspective: This study shows changes in ASIC mRNA expression and behavioral hyperalgesia of C57Bl/6 (wild type), ASIC1-/-, and ASIC3-/- mice before and after the induction of muscle inflammation. A-317567 was effective in reversing hyperalgesia in these animals, suggesting the potential of ASICs as therapeutic targets for muscle inflammatory pain.

Figure 1

Comparative qRT-PCR analysis for ASIC1, ASIC2, and ASIC3 mRNAs of L4, L5, and L6 DRGs from C57Bl/6 versus C57Bl/6-inflamed mice. There are bilateral increases in ASIC2 and ASIC3, and not ASIC1 mRNA levels, 24 hours after muscle inflammation. No differences were measured between the ipsilateral and contralateral mRNAs. (* = significantly greater than uninflamed mice, P<0.05).

Figure 2

Comparative qRT-PCR analysis for ASIC1, ASIC2, and ASIC3 mRNAs in L4, L5, and L6 DRGs from C57Bl/6, C57Bl/6-inflamed, ASIC3−/− inflamed, and ASIC1−/− inflamed mice. ASIC2 mRNA was increased in C57Bl/6-inflamed, ASIC3−/− inflamed and ASIC1−/− inflamed mice. ASIC3 mRNA increased in C57Bl/6-inflamed and in ASIC1−/− inflamed mice. ASIC1 mRNA levels were not significantly different between the groups. (* = significantly increased from C57Bl/6 uninflamed mice, P<0.05).

Figure 3

A and B) Muscle hyperalgesia (tweezers) develops similarly in both C57Bl/6 (WT) and ASIC3−/− mice after muscle inflammation on both ipsilateral and contralateral sides. ASIC1−/− mice do not develop muscle hyperalgesia after muscle inflammation. C and D) Cutaneous mechanical hyperalgesia (von Frey) develops similarly in both C57Bl/6 (WT) and ASIC1−/− mice on both sides. Cutaneous hyperalgesia does not develop in ASIC3−/− mice after muscle inflammation. (* = P<0.05).

Figure 4. Effect of A-317567 on muscle inflammation in C57Bl/6 (WT), ASIC1−/− and ASIC3−/− mice

A, C and E Muscle sensitivity (tweezers) in mice before and 24 h after carrageenan-induced muscle inflammation, and after A-317567 treatment. B, D and F Cutaneous mechanical sensitivity before and after muscle inflammation, and after A-317567 treatment. Closed symbols = ipsilateral, open symbol = contralateral sides. C57Bl/6 (WT) mice (A and B) develop muscle hyperalgesia (tweezers) (A) and cutaneous mechanical hyperalgesia (B) 24 h after carrageenan-induced muscle inflammation and is reversed by A-317567. Hyperalgesia and reversal with A-317567 is seen in both the ipsilateral and contralateral sides. ASIC1−/− mice (C and D), do not develop muscle hyperalgesia after muscle inflammation and A-317567 has no effect on muscle withdrawal thresholds (C). Cutaneous mechanical hyperalgesia develops in ASIC1−/− mice after muscle inflammation and A-317567 reverses the hyperalgesia on both sides (D). ASIC3−/− mice (E and F) develop muscle hyperalgesia after muscle inflammation and A-317567 reverses the effect on both ipsilateral and contralateral sides (E). A-317567 has no effect on mechanical responsiveness in ASIC3−/− mice that do not develop mechanical hyperalgesia after muscle inflammation (F). (* = P<0.05 when compared to the vehicle).