Electroacupuncture activated local sympathetic noradrenergic signaling to relieve synovitis and referred pain behaviors in knee osteoarthritis rats.

Introduction: Recent research has focused on the local control of articular inflammation through neuronal stimulation to avoid the systemic side effects of conventional pharmacological therapies. Electroacupuncture (EA) has been proven to be useful for inflammation suppressing and pain reduction in knee osteoarthritis (KOA) patients, yet its mechanism remains unclear. Methods: In the present study, the KOA model was established using the intra-articular injection of sodium monoiodoacetate (MIA) (1 mg/50 µL) into the knee cavity. EA was delivered at the ipsilateral ST36-GB34 acupoints. Hind paw weight-bearing and withdrawl thresholds were measured. On day 9, the histology, dep enrichment proteins, cytokines contents, immune cell population of the synovial membrane of the affected limbs were measured using HE staining, Masson staining, DIA quantitative proteomic analysis, flow cytometry, immunofluorescence staining, ELISA, and Western Blot. The ultrastructure of the saphenous nerve of the affected limb was observed using transmission electron microscopy on the 14th day after modeling. Results: The result demonstrated that EA intervention during the midterm phase of the articular inflammation alleviated inflammatory pain behaviors and cartilage damage, but not during the early phase. Mid-term EA suppressed the levels of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 in the synovium on day 9 after MIA by elevating the level of sympathetic neurotransmitters Norepinephrine (NE) in the synovium but not systemic NE or systemic adrenaline. Selective blocking of the sympathetic function (6-OHDA) and ß2-adrenergic receptor (ICI 118,551) prevented the anti-inflammatory effects of EA. EA-induced increment of the NE in the synovium inhibited the CXCL1-CXCR2 dependent overexpression of IL-6 in the synovial macrophages in a ß2-adrenergic receptor (AR)-mediated manner. Discussion: These results revealed that EA activated sympathetic noradrenergic signaling to control local inflammation in KOA rats and contributed to the development of novel therapeutic neurostimulation strategies for inflammatory diseases.

[Effect of electroacupuncture on spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage in rats with knee osteoarthritis].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on pain behavior, synovial inflammatory response and demyelination of saphenous nerve in the rats modeled with knee osteoarthritis (KOA) and explore the effect mechanism of EA for reliving allodynia. METHODS: Eighty-four male SD rats were randomly divided into a control group, a model group and an EA group, 28 rats in each one. Intra-articular injection of sodium monoiodoacetate (MIA) was administered in right knee joint of each rat in the model group and the EA group to establish the KOA model. In the EA group, separately, on day 5, 7 and 9 after modeling, EA was applied at "Zusanli" (ST 36) and "Yanglingquan" (GB 34) on the right side, with disperse-dense wave (2 Hz/15 Hz), 1 mA in current intensity, for 30 min in one intervention, once a day, and 3 interventions were required. On the 9th day after modeling, the weight-bearing rate was calculated for the affected limbs of the rats in each group, the synovial morphological changes were observed using HE and Masson staining, flow cytometry was adopted to detect the synovial immunocyte counts, and MSD multi-spot assay was used to detected the synovial inflammatory cytokine content. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was observed in each group and the ultrastructure of the saphenous nerve was observed under transmission electron microscopy. RESULTS: On the 9th day after modeling, compared with the control group, the weight-bearing rate of the affected limb was reduced (P<0.01), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis occurred in the affected limb; the counts of synovial CD11b+ cells and M1 macrophages (CD11b+CD86+) were increased (P<0.01), the contents of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß, IL-10 and IL-13 in the synovial tissue were elevated (P<0.01, P<0.05) for the rats of the model group. Compared with the model group, the weight-bearing rate of the affected limb was increased (P<0.05), the synovial hyperplasia, inflammatory cell infiltration and synovial fibrosis were mitigated, the counts of CD11b+ cells and M1 macrophages (CD11b+CD86+) in the synovial tissue, and the contents of TNF-α and IL-6 were reduced (P<0.01, P<0.05) in the EA group. On the 14th day after modeling, the hind-paw mechanical withdrawal threshold was reduced in the model group when compared with the control group (P<0.01), and it was increased in the EA group when compared with the model group (P<0.05). Besides, in the model group, obviously, the myelin sheath structure was destroyed, the myelin layer was disintegrated and loosened, the axon was extruded or the layer thicken and cracked. Compared with the model group, the injury of saphenous nerve was alleviated remarkably in the EA group. CONCLUSION: The intervention with EA may attenuate the synovial inflammatory response and the injury of saphenous nerve in the affected limb of the rat with KOA, so that the spontaneous pain during the synovial inflammatory response stage and allodynia at the later stage are relieved.

[Sensory and sympathetic nerves are involved in the changes of skin temperature, blood infusion and inflammatory cytokines of cutaneous tissue in the sensitized area of colitis rats].

OBJECTIVE: To investigate the changes of skin temperature, blood infusion and inflammatory cytokines of cutaneous tissue in the sensitized area of colitis model rats, as well as the relationship between sensory and sympathetic nerves and the formation of sensitized area, and to initially reveal the partial physical-chemical characteristics of the sensitized area in the colitis model rats. METHODS: Thirty-five male SD rats were randomly divided into a control group (n=10), a model group (n=18) and a guanethidine group (n=7). 5% dextran sulfate sodium (DSS) was adopted for 6-day free drinking to establish colitis model in the model group and the guanethidine group. On day 6 and 7, in the guanethidine group, guanethidine solution (30 mg/kg) was injected intraperitoneally for sympathetic block. On day 7, after injection of evans blue (EB) solution, the EB extravasation areas on the body surface were observed to investigate the distribution and physical-chemical characteristics of the sensitized area. The control area was set up, 0.5 cm away from the sensitized area, and with the same nerve segment innervation. Disease activity index (DAI) score of rats was compared between the normal group and the model group, and the morphological changes in the colon tissue were investigated with HE method. Using infrared thermal imaging technology and laser speckle flow imaging technology, skin temperature and blood infusion were determined in the sensitized area and the control area of the rats in the model group. Immunofluorescence technique was adopted to observe the expression levels of the positive nerve fibers of substance P (SP), calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH), and the correlation with blood vessels; as well as the expression levels of SP positive nerve fibers/tryptase+ mast cells, and tryptase+ mast cells/5-hydroxytryptamine (5-HT) in skin tissue in the sensitized area and the control area of the rats in the model group. MSD multi-level factorial method and ELISA were applied to determine the contents of pro-inflammatory and anti-inflammatory cytokines (e.g. TNF-α, IL-1ß, IL-6, IL-4 and IL-10) and anti-inflammatory substance corticosterone (CORT). RESULTS: Sensitization occurred at the T12-S1 segments of the colitis model rats, especially at L2-L5 segments. Compared with the normal group, DAI score was increased in the rats of the model group (P<0.05), and the colonic mucosal damage was obvious, with the epithelial cells disordered, even disappeared, crypt destructed, submucosal edema and a large number of inflammatory cells infiltrated. In comparison with the control area, the skin temperature and blood infusion were increased in the sensitized area of the model group (P<0.05, P<0.01); as well as the expression levels of the positive nerve fibers of SP, CGRP and TH of skin tissue (P<0.05), which was specially distributed in peripheral vessels, the expression levels of SP positive nerve fibers/tryptase+ mast cells, and tryptase+ mast cells/5-HT of the skin tissue were all expanded (P<0.05) in the sensitized area of the model group. Compared with the model group, the number of sensitized areas was reduced in the guanethidine group (P<0.05). In comparison with the control area of the model group, in the sensitized area, the contents of pro-inflammatory cytokines, e.g. TNF-α, IL-1ß and IL-6, and the anti-inflammatory substance CORT of skin tissue were all increased (P<0.05); and the contents of IL-6 and TNF-α were negatively correlated with CORT (P<0.05). CONCLUSION: The sensitized areas on the body surface of colitis rats are mainly distributed in the L2-L5 segments. Sensory and sympathetic nerves are involved in the acupoint sensitization, and the sensitized areas may have the dynamic changes in pro-inflammatory and anti-inflammatory substances.