The role of alpha5 nicotinic acetylcholine receptors in mouse models of chronic inflammatory and neuropathic pain

Abstract

The aim of the present study was to determine the impact of α5 nicotinic acetylcholine receptor (nAChR) subunit deletion in the mouse on the development and intensity of nociceptive behavior in various chronic pain models. The role of α5-containing nAChRs was explored in mouse models of chronic pain, including peripheral neuropathy (chronic constriction nerve injury, CCI), tonic inflammatory pain (the formalin test) and short and long-term inflammatory pain (complete Freund's adjuvant, CFA and carrageenan tests) in α5 knock-out (KO) and wild-type (WT) mice. The results showed that paw-licking time was decreased in the formalin test, and the hyperalgesic and allodynic responses to carrageenan and CFA injections were also reduced. In addition, paw edema in formalin-, carrageenan- or CFA-treated mice were attenuated in α5-KO mice significantly. Furthermore, tumor necrosis factor-alpha (TNF-α) levels of carrageenan-treated paws were lower in α5-KO mice. The antinociceptive effects of nicotine and sazetidine-A but not varenicline were α5-dependent in the formalin test. Both hyperalgesia and allodynia observed in the CCI test were reduced in α5-KO mice. Nicotine reversal of mechanical allodynia in the CCI test was mediated through α5-nAChRs at spinal and peripheral sites. In summary, our results highlight the involvement of the α5 nAChR subunit in the development of hyperalgesia, allodynia and inflammation associated with chronic neuropathic and inflammatory pain models. They also suggest the importance of α5-nAChRs as a target for the treatment of chronic pain.

Keywords: Inflammatory pain; Neuropathic pain; Nicotinic acetylcholine receptors; alpha5.

Fig. 1. Pain behavior and paw edema in α₅ KO and WT mice in the formalin test

The paw licking response after intraplantar injection of (A) 2.5% formalin concentration into the right paw of both α₅ KO and their WT littermate mice. Changes in paw edema (B), as measured by the difference in the ipsilateral paw diameter before and after injection (ΔPD), in α₅ WT and KO mice 1 hour after intraplantar injection of formalin. Data were given as the mean ± S.E.M. of 8 animals for each group. *p<0.05 significantly different from WT group.

Fig. 2. Effect of nicotine, varenicline and sazetidine-A in α₅ KO and WT mice in the formalin test

The effects of subcutaneous administration of nicotine (1.5 mg/kg), varenicline (3 mg/kg) and sazetidine-A (1.5 mg/kg) before intraplantar formalin (2.5 %) injection on paw licking time at phase I (A) and phase II (B) in the formalin test. Data were given as the mean ± S.E.M. of 8 animals for each group. *p<0.05 significantly different from corresponding vehicle and #p< 0.05 significantly different from corresponding WT group.

Fig. 3. Carrageenan-induced hyperalgesia, allodynia, edema and increase in TNF-α paw levels in α₅ KO and WT mice

Differences in paw withdrawal latencies (Δ PWL=contralateral–ipsilateral hindpaw latencies) (A) and mechanical paw withdrawal thresholds (PWT) (B) in α₅ KO and their WT littermate mice different times after intraplantar injection of carrageenan (0.5 % solution/20 μl). Degree of edema (C), as measured by the difference in the ipsilateral paw diameter before and after injection (ΔPD), in α₅ KO and WT mice 6 hrs after intraplantar injection of carrageenan. Effect of carrageenan on TNF-α paw levels (D) at 6 hours after intraplantar injection of carrageenan in α₅ KO and WT mice. Data were expressed as the mean ± S.E.M. of 6 animals for each group. *p<0.05 significantly different from the value of saline treated WT group, #p<0.05, significantly different from the value of carrageenan treated WT group. BL: baseline.

Fig. 4. CFA-induced hyperalgesia, allodynia and edema in α₅ KO and WT mice

Differences in paw withdrawal latencies (Δ PWL=contralateral–ipsilateral hindpaw latencies) (A) and mechanical paw withdrawal thresholds (PWT) (B) in α₅ KO and their WT littermate mice different times after intraplantar injection of CFA (75 and 50% solution/20 μl, respectively). Degree of edema (C), as measured by the difference in the ipsilateral paw diameter before and after injection of 75% CFA (ΔPD), in α₅ KO and WT mice days after intraplantar injection of CFA. Data were expressed as the mean ± S.E.M. of 5–8 animals for each group. *p<0.05 significantly different from WT group. BL: baseline

Fig. 5. Chrna5 (α₅ nAChR) deficiency impairs thermal hyperalgesia and mechanical allodynia responses in the injured sciatic nerve CCI model of neuropathic pain

Differences in paw withdrawal latencies (Δ PWL=contralateral–ipsilateral hindpaw latencies) (A) and paw withdrawal thresholds (PWT) (B) were determined in WT and α₅ KO mice at weekly different time points after chronic constrictive nerve injury (CCI) operation. Data were given as the mean ± S.E.M. of 6–10 animals for each group. *p<0.05 significantly different from the value of WT group. BL: baseline

Fig. 6. Altered anti-allodynic efficacy of nicotine in Chrna5 (α₅ nAChR) mutant mice

Nicotine (0.9 mg/kg, i.p.) reversed already-developed mechanical allodynia produced by CCI (week 7 post surgery) in the α₅ WT but not in KO mice. The mechanical paw withdrawal threshold of ipsilateral paw (A) and AUC of threshold values (B) are shown. Data were expressed as the mean ± S.E.M. of 6 animals for each group. *p<0.05 significantly different from test 0. #p<0.05 significantly different from corresponding value of WT group. BL: baseline.

Fig. 7. Anti-allodynic effects of supraspinal, spinal and/or peripheral nicotine in α₅ mutant mice

A head-to head comparison of supraspinal (25 μg/5μl, i.c.v.) (A), spinal (20 μg/5μl, i.t) (B) and peripheral (45 μg/20 μl, intraplantar) (C) nicotine anti-allodynia against CCI-induced neuropathic pain in α₅ WT and KO mice were tested using identical parameters at the peak of allodynia (14 days post-CCI). Data were expressed as the mean ± S.E.M. of 6 animals for each group. *p<0.05 significantly different from test 0. #p<0.05 significantly different from corresponding value of WT group. BL: baseline.

Fig. 8. Conditioned place aversion test in α₅ KO and WT mice

Intraperitoneal injection of 1.2% acetic acid induced conditioned place aversion as a negative affective component of pain. Data were given as the mean ± S.E.M. of 6–8 animals for each group. *p<0.05, compared to the vehicle-injected mice. #p<0.05, compared to the acetic acid-injected WT mice.