DNIC-mediated analgesia produced by a supramaximal electrical or a high-dose formalin conditioning stimulus: roles of opioid and alpha2-adrenergic receptors

Abstract

Background: Diffuse noxious inhibitory controls (DNIC) can be produced by different types of conditioning stimuli, but the analgesic properties and underlying mechanisms remain unclear. The aim of this study was to differentiate the induction of DNIC analgesia between noxious electrical and inflammatory conditioning stimuli.

Methods: First, rats subjected to either a supramaximal electrical stimulation or an injection of high-dose formalin in the hind limb were identified to have pain responses with behavioral evidence and spinal Fos-immunoreactive profiles. Second, suppression of tail-flick latencies by the two noxious stimuli was assessed to confirm the presence of DNIC. Third, an opioid receptor antagonist (naloxone) and an alpha2-adrenoreceptor antagonist (yohimbine) were injected, intraperitoneally and intrathecally respectively, before conditioning noxious stimuli to test the involvement of descending inhibitory pathways in DNIC-mediated analgesia.

Results: An intramuscular injection of 100 microl of 5% formalin produced noxious behaviors with cumulative pain scores similar to those of 50 microl of 2% formalin in the paw. Both electrical and chemical stimulation significantly increased Fos expression in the superficial dorsal horns, but possessed characteristic distribution patterns individually. Both conditioning stimuli prolonged the tail-flick latencies indicating a DNIC response. However, the electrical stimulation-induced DNIC was reversed by yohimbine, but not by naloxone; whereas noxious formalin-induced analgesia was both naloxone- and yohimbine-reversible.

Conclusions: It is demonstrated that DNIC produced by different types of conditioning stimuli can be mediated by different descending inhibitory controls, indicating the organization within the central nervous circuit is complex and possibly exhibits particular clinical manifestations.

Figure 1

Weighted pain score [24,25]of a formalin injection in the hind limb. (A) An i.m. injection of 100 μl of 5% (Fm5), 10% (Fm10), or 20% (Fm20) formalin in the anterior tibial muscle induced dose-dependent pain scores and a biphasic pain pattern similar to that of a subcutaneous plantar injection (2%, 50 μl, Fp). The Fm20 group had a lower pain score in the early phase and fewer flinch responses for the entire period compared to the Fp group. However, the Fp and Fm20 groups had similar highest pain scores in the late phase. (B) The Fm20 group showed no statistical difference in the cumulative pain score from that of the Fp group, indicating the muscular injection with 100 μl of 20% formalin resulted in a strong noxious reaction. Rat numbers: Fp = 9, Fm5 = 7, Fm10 = 6, Fm20 = 7. ** p < 0.01 vs. Fp; ⁺ p < 0.05, ⁺⁺ p < 0.01 vs. Fm5; one-way ANOVA with Bonferroni's post hoc test.

Figure 2

Rostrocaudal distribution of Fos-immunoreactive (Fos-ir) neurons after two conditioning noxious stimulations. Fos-ir neurons were identified at the L2 (A, C, E, G) and L5 (B, D, F, H) spinal dorsal horn at 90 min after the E20 and Fm20 stimulations. The groups shown in the figure are: the E20 (A, B), E50 (C, D), Fm20 (E, F), and control groups (G, H). Fos-ir neurons were few in the control and E20 rats at all segments (A, B, G, H). Formalin (Fm20) and supramaximal electrical (E50) stimulation induced Fos expression in L2 and L5 superficial laminae (C-F). In comparison, E50-induced Fos-ir neurons were densely distributed at the medial one-third of the L2 superficial dorsal horn (C), whereas Fm20-induced expression was relatively loosely distributed in the superficial dorsal horns (E). Scale bar = 100 μm.

Figure 3

Numerical analysis of Fos-ir neurons at the side ipsilateral to the conditioning stimulus. Fos-labeled neurons were significantly higher in the Fm20 and E50 groups than in the control and E20 groups regardless of the spinal segment. No significant difference was found between the C and E20 groups, or between the Fm20 and E50 groups for all segments and laminae. Topographically, E50-induced Fos expression was higher in the higher segments (L2 and L3) than in the lower lumbar segments (L4 and L5). S, superficial laminae I/II; NP, nucleus proprius, laminae III/IV; D, deep laminae V; DH, dorsal horn. Rat numbers: C = 6, E20 = 6, E50 = 5, Fm20 = 6. * p < 0.05, ** p < 0.01 vs. C; ⁺ p < 0.05, ⁺⁺ p < 0.01 vs. E20; one-way ANOVA with Bonferroni's post hoc test.

Figure 4

Effect of noxious electrical and formalin stimulation-induced DNIC on the tail-flick latency. (A, B) Halothane-anaesthetized rats were grouped into sham needles (C), E10 (10× twitch intensity), E20, and E50 (> 50×) electrical stimulation of the right ST36 acupoint. Changes in tail-flick latencies were compared by the "maximal possible effect". Control rats exhibited consistent tail-flick latencies without anesthetic influence. The electrical stimuli produced intensity-dependent analgesia on the tail reflex and showed maximal DNIC effects within the stimulation period. Notably, E50 elicited a strong and long analgesic effect (B). (C, D) A high-dose formalin injection (Fm20) caused a distinct DNIC pattern from E50 stimulation in tail-flick suppression. However, total pain summation (area under the curve) indicated no statistical difference between E50 and Fm20 (D). The horizontal thick bar indicates the electrical stimulating period. Rat numbers: C = 11, E10 = 10, E20 = 11, E50 = 9, Fm20 = 10. * p < 0.05, *** p < 0.001 vs. C; ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 vs. E10; ^# p < 0.05, ^### p < 0.001 vs. E20; one-way ANOVA with Bonferroni's post hoc test.

Figure 5

Contribution of the opioidergic pathway to DNIC. The opioid receptor was antagonized by a 2 mg/kg intraperitoneal naloxone injection at time point 15 and 1 mg/kg at time point 30. Naloxone itself did not alter the tail-flick latency in the control group (A, the C+Nal line). (A, B) The E50-induced DNIC was not reversed by naloxone administration, whereas the Fm20-induced DNIC was reversed in both the early and late phases by time point-to-point comparisons (C) or by total pain summation (D). Veh, saline; Nal, naloxone. The horizontal thick bar indicates the electrical stimulating period. Rat numbers: E50+Veh = 9, E50+NAL = 7, C+NAL = 9, Fm20+Veh = 10, Fm20+Nal = 9. ** p < 0.01, *** p < 0.001 vs. C+Nal; ^# p < 0.05 vs. Fm20+Nal; one-way ANOVA with Bonferroni's post hoc test.

Figure 6

Contribution of the α2-adrenergic pathway to DNIC. The α2-adrenergic receptor was antagonized by an intrathecal yohimbine injection of 30 μg in 20 μl of saline, 15 min before the conditioning stimulus. Yohimbine did not alter the tail-flick latency in the control group (A, C+Yoh line). Unlike naloxone, yohimbine had the ability to reverse the DNIC effect produced by both E50 (A, B) and Fm20 (C, D). Veh, saline; Yoh, yohimbine. Rat numbers: E50+Veh = 9, E50+Yoh = 10, C+Yoh = 10, Fm20+Veh = 9, Fm20+Yoh = 9. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. C+Yoh; ^# p < 0.05, ^## p < 0.01 vs. E50+Yoh (A, B) or vs. Fm20+Veh (C, D), respectively; one-way ANOVA with Bonferroni's post hoc test.

Figure 7

A scheme of the proposed DNIC circuitry activated by noxious electrical or formalin stimulations. Both noxious stimuli at the hindlimb can excite projection neurons in the corresponding spinal dorsal horn (DH) to activate descending inhibitory systems (e.g. noradrenergic pathway, NA) in the supraspinal structures to inhibit the noxious excitability in different spinal segments, e.g. the tail. In comparison, noxious formalin and electroacupuncture (EA), which is a low-intense electrical stimulation and may not be DNIC-mediated, produces analgesia through NA- and opioid-dependent actions (left upper panel), whereas the analgesic effect of noxious electrical stimulus may not depend on activation of opioid receptors (opioid-independent, right upper panel). Symbols +: excitation; -: inhibition.