| Acupuncture has been used in clinical practice for treating a wide variety of medical conditions. Many studies have supported the efficacy of acupuncture for relieving pain and regulating the functions of internal organs and the endocrine system through activation of the opioid pathway and the autonomic nerve system. However, little is known in regards to local responses to acupoint stimulation. Acupuncture elicits many physiologic responses through stimulating nerve fibers in the skin and muscle, which can be deleted by blocking or cutting the nerves in the acupoints.Recent studies also indicate that mast cells are involved in initiating acupuncture signals through stimulating peripheral sensory nerves. Mast cell-neuron interaction contributes to the modulation of signal transmission pathways. A model of mast cell-neuron interaction at acupoint has been proposed to study Ca2+ signaling and ATP release in mast cells and neurons and applied to investigate the coupled response of mast cells and the nerve system to mechanical stimuli. Although more and more substances and cells have been recognized for their involvement in the effects of acupuncture, what is being stimulated at the acupoint is still unknown. The definite relationship between the nerve fiber and mast cell remains a question, especially during and after acupuncture stimulation. In order to investigate the result of acupuncture on the nerve fiber and mast cell in local acupoint areas, we chose to stimulate a commonly used acupoint-Hegu (LI4)-with MA. The local tissue was examined by fluorescent histochemistry and immunohistochemistry simultaneously, in which we exposed sensory nerve fibers using substance P (SP) and CGRP, mast cells and its associated chemical elements using tryptase, histamine (HA), and serotonin (5-HT), and blood vessel using phalliodin.Objectives:Previous studies have shown that the effects of manual acupuncture (MA) are contributed by collagen fibers and mast cells in local acupoints, at which acupuncture stimulation causes various afferent fiber groups to be excited. However what happens in local nerve fibers and mast cells after MA remains unclear. The aim of this study was to examine the response of cutaneous nerve fibers and mast cells to MA stimulation in acupoint. The contralateral acupoin of the same rat was used as a non-stimulated control. Iminnohistochemistry analysis were carried out to observe the expression of histamine (HA), serotonin (5-HT) and nociceptive neuropeptides, calcitonin gene-related peptide (CGRP) and substance P (SP), in the acupoin area. Mast cells were labeled with anti-mast cell tryptase antibody and simultaneously with HA or 5-HT primary antibodies to observe their co-expression. To address these issues, we would investigate:(1) the corresponding innervations with SP-and CGRP-positive nerve fibers, and (2) morphological and chemical changes of local mast cells at acupoint LI4 after MA.Methods:After MA stimulation, the anaesthetized rats were immediately perfused transcardially with 100 mL of 0.9% saline, followed by 300 mL of 4% paraformaldehyde in 0.1 M phosphate buffered solution (PBS, pH 7.4). After perfusion, the skin tissue in the area of LI4 (about 43 mm2) was dissected out from the dorsal fore-foot and stored in 25% sucrose PB at 4 8C. A series of skin sections from LI4 of MA and control rats were cut at a thickness of 20 mm on a cryostat (Thermo, Microm International FSE, Germany) and divided into five groups for the five sections. The sequentially mounted slides were prepared for their respective types of fluorescent immunohistochemical and histochemical staining. Primary antibodies, including mouse monoclonal anti-mast cell tryptase antibody (1:1000, Abeam, Hong Kong), rabbit polyclonal anti-mast cell tryptase antibody (1:500, Abeam), rabbit polyclonal anti-SP antibody (1:500, Abeam), mouse monoclonal anti-CGRP antibody (1:500, Abcam), rabbit polyclonal anti-HA antibody (1:500, Abeam), and rabbit polyclonal anti-5-HT antibody (1:500, Abcam) were used in this study. Goat anti-mouse Alexa Fluor 488 or 594 secondary antibody (1:500, Molecular Probes, Eugene, Oregon, USA) and goat anti-rabbit Alexa Fluor 488 or 594 secondary antibody (1:500; Molecular Probes) were used to visualize the corresponding primary antibodies. Additionally, Alexa Fluor 488 phalliodin (1:1000, Molecular Probes) and 40,6-diamidino-2-phenylindole dihydrochloride (DAPI,1:500000; Molecular Probes) were applied for conterstaining.The five groups of tissue sections around LI4 were treated with fluorescent immunohistochemical and histochemical stains to examine the relationship of:(1) mast cells and blood vessels, (2) mast cells and SP-positive nerve fibers, (3) mast cells and CGRP-positive nerve fibers, (4) HA-expression on mast cells, and (5) 5-HT-expression on mast cells.Samples were recorded with a confocal imaging system (FV1200, Olympus, Japan) and analyzed using the Olympus Image Processing Software.10 images were collected in successive frames of 2 mm from each section at the thickness of 20 mm (Z series) and integrated into a single in-focus image. Final images were processed with Adobe photoshop CS5 and Adobe illustrator CS5 (Adobe Systems, San Jose, CA, USA). Some of the selected images were used for analysis with three-dimensional reconstruc-tion using Imaris7.6.2 software (www.bitplane.com). The number of mast cells was recorded within a magnified field (400) in 10 randomized sections from each rat. Twenty randomized sections were analyzed with the Olympus Image Processing Software for quantifying intensity of fluorescence. Data was expressed as mean standard deviation and processed with the statistical software SPSS 16.0.3.Results:1.The results showed that:The acupoints in different locations such as Baihui (GV 20), Weishu (BL21), Zhongwan (RN12) and LI4 had the same constituent but the contents were different.2.The results showed that:1) mast cells migrated toward the blood vessels in the lower dermis and subcutaneous tissue and aggregated toward the site of needling after MA stimulation; 2) SP-and CGRP-positive nerve fibers responded to MA stimulation, displaying higher expression of SP and CGRP than those of the control, and, in turn, mast cells migrated toward SP-and CGRPpositive nerve fibers; 3) The aggregated mast cells released much more tryptase, HA, and 5-HT through degranulation after MA stimulation than those of the control.Conclusions:In summary, nerve fibers and mast cells in the area of acupoints respond to MA stimulation. MA stimulation induces high expression of nociceptive neuropeptides of SP and CGRP in the subepidermal nerve fibers, which then activate mast cells. Mast cells aggregate and degranulate, releasing tryptase,5-HT and HA. These neuroactive components play a role in conveying acupuncture signals to certain pathways to deliver the effects of acupuncture. |
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