Mast cell deficiency attenuates acupuncture analgesia for mechanical pain using c-kit gene mutant rats

Abstract

Background: Acupuncture therapy plays a pivotal role in pain relief, and increasing evidence demonstrates that mast cells (MCs) may mediate acupuncture analgesia. The present study aims to investigate the role of MCs in acupuncture analgesia using c-kit gene mutant-induced MC-deficient rats.

Materials and methods: WsRC-Ws/Ws rats and their wild-type (WT) littermates (WsRC-+/+) were used. The number of MCs in skin of ST36 area was compared in two rats after immunofluorescence labeling. Mechanical withdrawal latency (MWL), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were measured on bilateral plantar for pain threshold evaluation before and after each stimulus. Acupuncture- and moxibustion-like stimuli (43°C, 46°C heat, 1 mA electroacupuncture [EA], 3 mA EA, and manual acupuncture [MA]) were applied randomly on different days.

Results: Fewer MCs were observed in the skin of ST36 in mutant rats compared to WT rats (P<0.001). For pain thresholds, MWL and MWT were higher in WsRC-Ws/Ws compared to WsRC-+/+ on bilateral paws (P<0.05), but TWL was not different between the two rats (P>0.05). Bilateral MWL and MWT in WsRC-+/+ rats increased significantly after each stimulus compared to baseline (P<0.01, P<0.001). In WsRC-Ws/Ws rats, only noxious stimuli could produce anti-nociceptive effects for mechanical pain (46°C, 3 mA EA, MA) (P<0.01, P<0.001). Additionally, the net increases in MWL and MWT induced by most stimuli were greater in WT than in mutant rats (P<0.05). For thermal nociception, either high- or low-intensity stimuli could significantly augment TWL in two rats (P<0.001), and the net increases of TWL evoked by most stimuli were to the same extent in two genetic variants.

Conclusion: MCs influence the basic mechanical but not thermal pain threshold. MCs participate in acupuncture analgesia in mechanical but not in thermal nociception, in that MC deficiency may attenuate the mechanical analgesia evoked by high-intensity stimuli and eliminate analgesia provoked by low-intensity stimuli.

Keywords: WsRC-Ws/Ws rats; mechanical withdrawal threshold; stimulus intensity; thermal withdrawal latency; tryptase.

Figure 1

Immunofluorescence staining for tryptase (MCs) in ST36 skin tissue. Notes: Representative images of MC staining in WsRC-+/+ (A, a) and WsRC-Ws/Ws (B, b) rats (n=5). (C) MC count in mutant rats was significantly less than that of WT rats. Data were analyzed using independent t-test and expressed as mean ± standard error of the mean. ***P<0.001, vs WsRC-+/+ rats. Abbreviations: MCs, mast cells; WT, wild-type.

Figure 2

Baseline MWL, MWT, and TWL in left and right hind paws in WsRC-+/+ and WsRC-Ws/Ws rats. Notes: Baseline MWL (A) and MWT (B) in WT and mutant rats (n=15). (C) Baseline TWL in two rats. Data were analyzed using independent t-test and expressed as mean ± standard error of the mean. *P<0.05, compared with WsRC-+/+ rats. WsRC-Ws/Ws rats showed a longer MWL and higher MWT (P<0.05) than WT rats. Abbreviations: MWL, mechanical withdrawal latency; MWT, mechanical withdrawal threshold; TWL, thermal withdrawal latency; WT, wild-type.

Figure 3

MC deficiency blocked the effect of low-intensity stimuli on mechanical pain sensation. Notes: Baseline MWL and MWT data after different stimuli at 43°C and 46°C heat, 1 mA and 3 mA EA, and MA were collected in plantar test. Ipsilateral (A) and contralateral (B) paw MWL were increased by acupuncture- and moxibustion-like stimulation, either low or high intensities in WT rats, whereas only high intensity of stimulus increased ipsilateral and contralateral paw MWL in mutant rats. Ipsilateral (C) and contralateral (D) paw MWT were increased by acupuncture- and moxibustion-like stimulation, either low or high intensities in WT rats, whereas only high intensity of stimulus increased ipsilateral and contralateral paw MWL and MWT in mutant rats. Data were analyzed using one-way analysis of variance and least significant difference for post hoc analysis. *P<0.05, **P<0.01, ***P<0.001, compared with baseline MWL or MWT in each group. Data represents the mean ± standard error of the mean. Abbreviations: Contra., contralateral; EA, electroacupuncture; Ipsi., ipsilateral; MA, manual acupuncture; MC, mast cell; MWL, mechanical withdrawal latency; MWT, mechanical withdrawal threshold; WT, wild-type.

Figure 4

MC deficiency impaired the effect of acupuncture and moxibustion-like stimulation on the improvement of MWL and MWT. Notes: Data are represented as net increases of MWL and MWT (subtracting the baseline value from poststimulus values). Net increases of MWL in ipsilateral (A) and contralateral (B) paws in WsRC-+/+ and WsRC-Ws/Ws rats (n=15). Net increases of MWT in ipsilateral (C) and contralateral (D) paws in WsRC-+/+ and WsRC-Ws/Ws rats (n=15). Independent t-tests, *P<0.05, **P<0.01, vs WsRC-+/+ rats. MC deficiency significantly decreased most effects of acupuncture and moxibustion-like stimuli on MWL and MWT in WsRC-Ws/Ws rats as compared to those in WsRC-+/+ rats. Data are represented as mean ± standard error of the mean. Abbreviations: Contra., contralateral; Ipsi., ipsilateral; MC, mast cell; MWL, mechanical withdrawal latency; MWT, mechanical withdrawal threshold; EA, electroacupuncture; MA, manual acupuncture..

Figure 5

MC deficiency had no effect on TWL improvement induced by acupuncture and moxibustion stimuli. Notes: Ipsilateral (A) and contralateral (B) hind paw TWL of WsRC-+/+ and WsRC-Ws/Ws rats were increased by different intensities of acupuncture and moxibustion stimuli (n=15). All data were analyzed using one-way analysis of variance and least significant difference for post hoc analysis. ***P<0.001, compared with baseline TWL. Data are expressed as mean ± standard error of the mean. Abbreviations: Contra., contralateral; Ipsi., ipsilateral; TWL, thermal withdrawal latency; EA, electroacupuncture; MA, manual acupuncture; MC, mast cell.

Figure 6

MC deficiency did not influence the effect of acupuncture and moxibustion on TWL increase. Notes: Data are represented as net increase of TWL (subtracting the baseline value from poststimulus values). Net increase of TWL in ipsilateral (A) and contralateral (B) paws in WsRC-+/+ and WsRC-Ws/Ws rats (n=15). All data were analyzed using independent t-test and expressed as mean ± standard error of the mean. *P<0.05, compared with WsRC-+/+ rats. All improvements of TWL were similar in WT and mutant rats except the effect of 3 mA EA, that the increase of TWL in WT rats was significantly greater than that in mutant rats (P<0.05). Abbreviations: Contra., contralateral; EA, electroacupuncture; Ipsi., ipsilateral; TWL, thermal withdrawal latency; WT, wild-type; MA, manual acupuncture; MC, mast cell.