Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 18 (1), 74

Repeated Electroacupuncture Treatment Attenuated Hyperalgesia Through Suppression of Spinal Glial Activation in Chronic Neuropathic Pain Rats

Affiliations

Repeated Electroacupuncture Treatment Attenuated Hyperalgesia Through Suppression of Spinal Glial Activation in Chronic Neuropathic Pain Rats

Jun-Ying Wang et al. BMC Complement Altern Med.

Abstract

Background: Cumulated evidence reveals that glial cells in the spinal cord play an important role in the development of chronic neuropathic pain and are also complicated in the analgesic effect of EA intervention. But the roles of microgliacytes and astrocytes of spinal cord in the process of EA analgesia remain unknown.

Methods: A total of 120 male Wistar rats were used in the present study. The neuropathic pain model was established by chronic constrictive injury (CCI) of the sciatic nerve. The rats were randomly divided into sham group, CCI group, and sham CCI + EA group, and CCI + EA group. EA was applied to bilateral Zusanli (ST36)-Yanlingquan (GB34). The mechanical (both time and force responses) and thermal pain thresholds (PTs) of the bilateral hind-paws were measured. The number of microgliacytes and activity of astrocytes in the dorsal horns (DHs) of lumbar spinal cord (L4-5) were examined by immunofluorescence staining, and the expression of glial fibrillary acidic protein (GFAP) protein was detected by western blot.

Results: Following CCI, both mechanical and thermal PTs of the ipsilateral hind-paw were significantly decreased beginning from the 3rd day after surgery (P < 0.05), and the mechanical PT of the contralateral hind-paw was considerably decreased from the 6th day on after surgery (P < 0.05). CCI also significantly upregulated the number of Iba-1 labeled microgliacytes and the fluorescence intensity of glial fibrillary acidic protein (GFAP) -labeled astrocyte in the superficial laminae of DHs on bilateral sides (P < 0.05). After repeated EA, the mechanical and thermal PTs at bilateral hind-paws were significantly relieved (P < 0.05). The increased of number of microgliacytes was markedly suppressed by 2 days' EA intervention, and the average fluorescence intensity was suppressed by 2 weeks' EA. The expression of GFAP protein were down-regulated by 1 and 2 weeks' EA treatment, respectively (P < 0.05).

Conclusions: Repeated EA can relieve neuropathic pain and mirror-image pain in chronic neuropathic pain rats, which is probably associated with its effect in downregulating glial cell activation of the lumbar spinal cord, the microgliacyte first and astrocyte later.

Keywords: Astrocytes; Chronic neuropathic pain; Electroacupuncture; Lumbar spinal cord; Microgliacytes.

Conflict of interest statement

Ethics approval

All experimental procedures were approved by the Institute of Acupuncture and Moxibustion of China Academy of Chinese Medical Sciences and were identical to those recommended in the Guidelines for Laboratory Animal Care and Use from the Chinese Ministry of Science and Technology(2006).

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Repeated EA treatment reduced CCI-induced mechanical and thermal hyperalgesia and mirror-image pain in neuropathic pain rats (mean ± SD, n = 12/group). Both mechanical (response time: a, force threshold: b) and thermal paw withdrawal latencies (PWLs) at the ipsilateral (Ipsi., a, c, e) and contralateral (Cont., b, d, f) paws were observed before and every 3 days after chronic constrictive injury (CCI) of the sciatic nerve. Compared with the CCI rats, CCI-decreased mechanical PWLs were significantly increased by EA treatment from the 12th day on (time response: a) and 9th day on (force response: c) after surgery on the Ipsi. side (a, c), and from the 6th day on the Cont. side (b, d); and CCI-decreased thermal PWLs were significantly up-regulated by EA from the 6th day on after surgery on the Ipsi. side (e) and turned to the normal level from the 6th day on after surgery on the Cont. side (f), suggesting a positive effect of EA on chronic neuropathic pain and mirror-image pain. (*P < 0.05, vs the sham group; # P < 0.05, vs the CCI group)
Fig. 2
Fig. 2
Numbers of Iba-1-labeled microgliacytes in ipsilateral and contralateral dorsal horns (DHs) of lumbar spinal cord (L4–5) on day 6 and 18 after surgery in the normal control (normal), sham operation (sham), and CCI model (CCI) groups. a Iba-1-labeled microglia (indicated by arrowheads) in the DHs and anterior horns of lumbar spinal cord on the ipsi. Side of CCI on day 6 after surgery. b An increase of numbers of the activated microgliacytes mainly distributing in the superficial layers of ipsi. and cont. DHs on day 6 (denser) and day 18 (lesser) in the 3 groups. Central column: the magnified activated microgliacytes derived from the dashed rectangular box of each picture on the left side. Ipsi: ipsilateral, Cont: contralateral. D: day. c Histograms showing the number of Iba-1-labeled microgliacytes in the ipsilateral and contralateral DHs of lumbar spinal cord on day 6 and 18 after sham and CCI-operation in the 3 groups (mean ± SD, n = 6/group). ^ P < 0.05, ^^ P < 0.01, vs the normal group; # P < 0.05, ## P < 0.01, vs the sham group
Fig. 3
Fig. 3
Immunofluorescent staining of glia fibrillary acidic protein (GFAP) for displaying astrocytes in Ipsi and Cont DHs of lumbar spinal cord on day 6 and 18 after CCI in rats of the five groups. a Representative samples of immunofluorescence staining showing an increase of the number of GFAP-labeled astrocytes in the superficial layers of DHs of lumbar spinal cord on the Ipsi and Cont sides of surgery on day 18 (relevant to day 6). In the right column of both Ipsi and Cont DH section samples, a magnification of GFAP-labeled astrocytes (got from the dashed square boxes of the tissue section samples on the left side) is shown, suggesting an increase of number of the activated astrocytes after CCI. b Bar graphs showing the mean immunofluorescence intensity of GFAP in the Ipsi and Cont DHs on day 6 and 18 in the 5 groups. Eight rats in each group were examined and data are expressed in Mean ± SD(n = 6/group). ^^ P < 0.01, vs the normal group; # P < 0.05, ## P < 0.01, vs the sham group
Fig. 4
Fig. 4
Immunofluorescence staining showing activities of microgliacytes and astrocytes in Ipsi and Cont DHs of lumbar spinal cord on day 6 and 18 after CCI in rats. a Representative tissue sections of immunofluorescence staining showing Iba-1-labeled microgliacytes (red) and GFAP-labeled astrocytes (green) in the Ipsi DHs of the lumbar spinal cord. In the superior-right corner of each section, the magnified microgliacyte and astrocyte from each dashed square box are shown. b Bar graphs showing the numbers of microgliacytes and mean fluorescence intensity values of GFAP (for astrocytes) in the Ipsi DHs on day 6 and 18 in the sham and EA groups(mean ± SD, n = 6/group). Results showed that in the early period of CCI-induced neuropathic pain, 2 days’ EA suppressed the activation of microgliacytes (not astrocytes), and in the later period, 2 weeks’ EA suppressed the activation of astrocytes. # P < 0.05, vs the CCI group)
Fig. 5
Fig. 5
Representative section samples of dual immunofluorescent staining of Iba-1 (red) and neuronal nuclei (NeuN, green) for microgliacytes and neurons in the Ipsi ventral horns of lumbar spinal cord in the sham, CCI 6D and 18D, and CCI + EA2W groups. The images from the left to the right longitudinal rows are Iba-1-labeled microgliacytes, Iba-1 and NeuN labeled cells and magnified cells chosen from the dashed square box of each image on their individual left side. The NeuN-positive neurons were closely surrounded by many microgliacytes after CCI, suggesting a remodeling of the lumbar locomotor circuitry. NeuN, a neuronal specific nuclear protein and a biomarker for neurons
Fig. 6
Fig. 6
Quantitative analysis of expression levels of GFAP protein in the dorsal part of the lumbar spinal cord in rats of the five groups. Upper panel: representative western blot stripes of the 5 groups. 1: control, 2: CCI(18D), 3: CCI + EA2D, 4; CCI + EA1W, 5:CCI + EA2W. Lower panel: bar graphs showing the expression levels of GFAP protein in the 5 groups, five rats in each group were examined and the data are expressed as mean ± SD. * P < 0.05, vs the control group; # P < 0.05, vs the CCI group

Similar articles

See all similar articles

References

    1. Gao YJ, Ji RR. Targeting astrocyte signaling for chronic pain. Neurotherapeutics. 2010;7(4):482–493. doi: 10.1016/j.nurt.2010.05.016. - DOI - PMC - PubMed
    1. Hulsebosch CE. Special issue on microglia and chronic pain. Exp Neurol. 2012;234(2):253–254. doi: 10.1016/j.expneurol.2012.01.009. - DOI - PubMed
    1. Wang W, Wang W, Mei XP, Huang J, Wei Y, Wang Y, Wu S, Li Y. Crosstalk between spinal astrocytes and neurons in nerve injury-induced neuropathic pain. PLoS One. 2009;4(9):1–10. - PMC - PubMed
    1. Sfieh-Garabedian B, Poole S, Haddad JJ, Massaad CA, Jabbur SJ, Saade NE. The role of the sympathetic efferents in endotoxin- induced localized inflammatory hyperalgesia and cytokine upregulation. Neuropharmacology. 2002;42(6):864–872. doi: 10.1016/S0028-3908(02)00028-X. - DOI - PubMed
    1. Vallejo R, Tilley DM, Vogel L, Benyamin R. The role of glia and the immune system in the development and maintenance of neuropathic pain. Pain Practice. 2010;10(3):167–184. doi: 10.1111/j.1533-2500.2010.00367.x. - DOI - PubMed

Substances

LinkOut - more resources

Feedback