Electroacupuncture Attenuates 5'-Guanidinonaltrindole-Evoked Scratching and Spinal c-Fos Expression in the Mouse

Abstract

The present study was undertaken to investigate the influence of electroacupuncture (EA) on compulsive scratching in mice and c-Fos expression elicited by subcutaneous (s.c.) administration of a known puritogen, 5'-guanidinonaltrindole (GNTI) to the neck. Application of EA to Hegu (LI4) and Quchi (LI11) acupoints at 2 Hz, but not 100 Hz, attenuated GNTI-evoked scratching. In mice pretreated with the µ opioid receptor antagonist naloxone, EA 2 Hz did not attenuate GNTI-evoked scratching, whereas EA at 2 Hz did attenuate GNTI-evoked scratching in mice pretreated with the κ opioid receptor antagonist nor-binaltorphimine. Moreover, intradermal (i.d.) administration of the selective µ opioid receptor agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate (DAMGO) attenuated GNTI-evoked scratching behavior, while s.c. administration of DAMGO was ineffective. GNTI provoked c-Fos expression on the lateral side of the superficial layer of the dorsal horn of the cervical spinal cord. Application of 2 Hz EA to LI4 and LI11 decreased the number of c-Fos positive nuclei induced by GNTI. It may be concluded that application of 2 Hz EA to LI4 and LI11 attenuates scratching behavior induced by GNTI in mice and that the peripheral µ opioid system is involved, at least in part, in the anti-pruritic effects of EA.

Figure 1

Photograph of a mouse under isoflurane anesthesia with acupuncture needles inserted at Hegu (LI4) and Quchi (LI11) acupoints. LI4 is located on the first dorsal interossei, radial to the midpoint of the second metacarpal bone in the forelimb. LI11 is located in the depression on the lateral end of the cubital crease in the forelimb, when the elbow is fully flexed [24].

Figure 2

Flow chart depicting steps involved in the EA study.

Figure 3

Effects of EA at 2 Hz and 100 Hz on GNTI- (0.3 mg/kg) induced scratching. EA was applied to the LI4 and LI11 acupoints before GNTI administration. The number of scratches was counted for 40 min after pruritogen injection. EA at 2 Hz, but not 100 Hz, attenuated GNTI-induced scratching behavior (*P < 0.05 compared to controls). Between-group comparisons for each group were performed by ANOVA, followed by Tukey's test.

Figure 4

Effects of EA at 2 Hz on GNTI- (0.3 mg/kg) induced scratching. EA was applied to the bilateral ST36 acupoints prior to GNTI administration. The number of scratches was counted for 40 min after GNTI injection. EA at 2 Hz had no effect upon GNTI-induced scratching behavior. Between-group comparisons for each group were performed by Student's t-test.

Figure 5

Effects of EA at 2 Hz on GNTI- (0.3 mg/kg) induced scratching in naloxone-pretreated mice. Naloxone (s.c.; 1 mg/kg) was administered 70 min before GNTI injection, that is, 5 min before isoflurane anesthesia for EA. EA was applied to the LI4 and LI11 acupoints before GNTI administration. The number of scratches was counted for a period of 40 min after pruritogen injection. EA failed to attenuate GNTI-induced scratching behavior in naloxone-pretreated mice. Between-group comparisons for each group were performed by Student's t-test.

Figure 6

Effects of EA at 2 Hz on GNTI- (0.3 mg/kg) induced scratching in nor-binaltorphimine-pretreated mice. Nor-binaltorphimine (10 mg/kg) was subcutaneously administered 24 hr before GNTI injection [25]. EA was applied to the LI4 and LI11 acupoints before GNTI administration. The number of scratches was counted for 40 min after pruritogen injection. EA attenuated GNTI-induced scratching behavior in nor-binaltorphimine-pretreated mice (*P < 0.05). Between-group comparisons for each group were performed by Student's t-test.

Figure 7

Effects of DAMGO by i.d. injection (a) and by s.c. injection (b) on GNTI- (0.3 mg/kg) induced scratching. (a) Mice were administered an i.d. injection of saline or DAMGO (10 nmol) at 15 min before GNTI (0.3 mg/kg). (b) Mice were administered a s.c. injection of saline or DAMGO (10 nmol) at 15 min before GNTI (0.3 mg/kg). The number of scratches was counted for 30 min after GNTI injection. DAMGO (10 nmol/site) attenuated GNTI-induced scratch behavior by i.d. administration but not s.c. administration. (*P < 0.05 compared to controls). Between-group comparisons for each group were performed by Student's t-test.

Figure 8

Flow chart depicting steps involved in the immunohistochemistry (IHC) study.

Figure 9

Representative photomicrographs of c-Fos expression in the spinal cord. c-Fos was not detected in the superficial layers of the dorsal horn following saline injection (a)–(c) and after EA (2 Hz) application at the LI4 and LI11 acupoints (d)–(f). (a)–(c) represent one spinal section, with increasing magnification, while (d)–(f) are from another spinal section, with increasing magnification.

Figure 10

Representative photomicrographs of c-Fos expression induced by GNTI in the spinal cord and the effects of EA. GNTI (0.6 mg/kg; s.c.; behind the neck) induced c-Fos expression on the lateral side of the superficial lamina of the dorsal horn of the cervical spinal cord without (a)–(c) and with (d)–(f) EA (2 Hz) pretreatment. (a)–(c) represent one spinal section, with increasing magnification, while (d)–(f) are from another spinal section, with increasing magnification.

Figure 11

Effects of EA on GNTI- (0.6 mg/kg) induced scratching behavior (a) and c-Fos expression (b). (a) Effects of EA 2 Hz (applied to the LI4 and LI11 acupoints) on GNTI- (0.6 mg/kg) induced scratches. The number of scratches was counted for 40 min after injection of GNTI. EA at 2 Hz reduced GNTI- (0.6 mg/kg) induced scratching behavior (*P < 0.05 compared to control by Student's t-test). (b) The numbers of c-Fos positive nuclei were counted and averaged from 12 randomly chosen cervical sections from each animal in each group. EA at 2 Hz was applied to the LI4 and LI11 acupoints before administration of GNTI (0.6 mg/kg, s.c.). EA (2 Hz) significantly decreased the number of c-Fos positive nuclei (*P < 0.05, Student's t-test; N: the number of animals).