Cutaneous neurogenic inflammation in the sensitized acupoints induced by gastric mucosal injury in rats
© The Author(s). 2017
Received: 10 September 2016
Accepted: 12 January 2017
Published: 7 March 2017
In acupuncture practice, the most important step is to confirm the location of a sensitized acupoint which reflects a diagnosis and can be stimulated with a specialized needle to treat the disease. Abnormal symptoms such as hyperalgesia or allodynia at the sensitized acupoints in patients with visceral disorders are considered to be in relation with referred pain and neurogenic inflammation. Yet, limited study has investigated the cutaneous neurochemical changes of the sensitized acuponits.
The resent study developed an animal model of gastric mucosal injury (GMI) by HCl administered into the stomach of the rats. Evans Blue (EB) dye was applied by injection of tail vein after mucosal damage to observe the neurogenic plasma extravasation dots in the skin of the rats. The EB dots extravagated in the skin were compared with locations of acupoints. Immnohistochemistry analysis was used to detect the expression of calcitonin gene-related peptide (CGRP)- or substance P (SP)-labeled nerve fibers, histamine (HA)-, serotonin (5-HT)-, and tryptase-labeled cells in EB dots. Images were recorded and analyzed by Confocal imaging system and Olympus Image Processing Software.
The results showed that GMI resulted in neurogenic plasma extravasation in the skin of the acupoints over the back and abdomen, which mostly occurred in the T9-11 dermatomere. The EB extravasation dots appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than that in regions beside EB dots and in the control, mostly distributed in the nerve fibers around both the vessels and root of hair follicle. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA around the vessels in areas of the EB dots.
Our results indicates that the mechanism of EB extravasation in the skin of the acupoints induced by GMI are closely related to neurogenic inflammation, and that the high expression of local allergic substances and nociceptive neuropeptides in the local skin including SP, CGRP, HA, 5-HT, and mast cell tryptase may be the underlying mechanism of the acupoint sensitization.
KeywordsSensitized acupoints Gastric mucosal injury Nociceptive neuropeptide HA 5-HT Mast cell
In Chinese acupuncture practice, doctors’ careful detection of acupoints and patients’ subjective response to acupoint stimulation are major factors in effective treatment. Clinically, the most important step is to confirm the location of a sensitized acupoint, which then reflects a diagnosis, and can be stimulated with a specialized needle for treating the disease. Abnormal symptoms such as hyperalgesia or allodynia occurred at some sensitized acupoints in most patients when they are suffered with visceral disorders . The sensitized points may be regular points, extra points, or “Ashi” points .
Hyperalgesia or allodynia at the sensitized acupoints are considered to be in relation with “referred pain”, a term defined by Head in 1983 and refers to the many forms of visceral pain felt in regions of the body other than the organ whose stimulation causes pain . Over the past three decades connections between Head zones and acupoints have been discussed [4, 5]. A comparison of Head’s papers with the oldest still extant Chinese sources on acupuncture revealed astonishing parallels between the two concepts regarding both point locations and functional aspects . Strong functional relations between all Head zones, channels, and acupoints were found when following the pattern of segmental dermatomes , that is, areas of skin innervated by one and the same spinal nerve.
Previous studies had suggested that mechanism of the referred visceral pain is related to central sensitization of the spinal cord and neurogenic inflammation [6, 7]. Recently,animal study on the mechanism of acupoint sensitization confirmed that pathological change of internal organs’ functional activity lead to the sensitization of spinal center and further to the changes of the size and function of acupoints on body surfaces . Yet, limited study has investigated the cutaneous neurochemical changes of the sensitized acuponits.
The purpose of this study was to develop an animal model of gastric mucosal injury (GMI) by acid-induced nociceptive event in rats. Evans Blue (EB) dye was applied by injection of tail vein after mucosal damage and the referred EB dots extravagated in the skin  were observed to give evidence for the kinetic distribution of sensitized acupoints. Immunohistochemistry analyses were used to observe the changes of mast cells (MCs), the expression of nociceptive neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), and allergic substances such as histamine (HA) and serotonin (5-HT) in the skin in the extravasated EB dots, to further investigate the underlying mechanisms.
Animals and model procedures
All experimental protocols reported here were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and the Animal Use and Care of Medical Laboratory Animals from the Ministry of Public Health of People’s Republic of China. All efforts were made to minimize the number of animals used and their suffering. Sprague-Dawley rats, weighing 180–220 g, were bought from the institute of Zoology, Chinese Academy of Sciences. The rats were housed in a room maintained at 24 ± 1 °C and illuminated for 12 h (07:00 to 19:00) every day, and fasted for 20 h prior to experiments but had free access to water. Thirty-six rats were randomly divided into six groups: HCl 1d group, HCl 2d group, HCl 3d group, HCl 4d group, HCl 5d group, and Saline control group, with 6 rats in each group. Hydrochloric acid (HCl, 0.5 M) or saline (as a control) was administered orally at a volume of 1 mL/100 g via a soft pediatric feeding tube . After modeling, thirty rats in all the HCl groups were used to observe the dynamic change of EB extravasated points in successive five days. After observation the EB extravasated points, the stomachs and the skin of the six rats in the HCl 1d group were used for immunochemistry experiments.
Evans blue dye injection and assessment of cutaneous plasma extravasation
Five to six hours after HCl administration, the rats were anesthetized with pentobarbital (50 mg/kg i.p.). EB Dye (Sigma Chemical Co, St. Louis, MO) dissolved in sterile water (50 mg/kg) was then injected through the tail vein. Skin color changes were observed for 1–2 h, and the area of dye extravasation was sketched on the body charts and, in some rats, photographed. Care was taken not to cause any injury to the skin before and during hair removal. The hair of the back, thorax, abdomen, arms, and legs was shaved and sometimes completely removed using commercially available hair removal cream.
Tissue preparation and immunohistochemical staining
Primary antibodies for immunohistochemistry
Anti-Mast Cell Tryptase
Data analysis and statistical analysis
EB dye extravasation was quantified by counting the number of blue dots in the skin over the same dermatomere areas. Data are expressed as mean ± SEM. The Kolmogorov-Smirnof test was used to evaluate if these groups fit the normal distributions. Normally distributed groups were analyzed by parametric tests. Comparison between three groups was made using the One-Way ANOVA test followed by the LSD or Dunnett’s T3 post-hoc test. Nonnormally distributed groups were analyzed by the Mann-Whitney test. P < 0.05 was considered significant.
GMI induced EB extravasated points in certain acupoints
Nociceptive neuropeptides CGRP and SP were highly expressed in EB extravasated dots
Mast cells aggregated and degranulated and released HA and 5-HT in EB extravasated dots
The results of the present study demonstrated that GMI resulted in neurogenic plasma extravasation in the skin of the acupoints over the back and abdomen, mostly in the T9-11 dermatomere. The EB extravasation dots appeared after GMI and disappeared gradually during the natural self-recovery of the gastric mucosa. More SP and CGRP positive nerve fibers were distributed in EB dots than in regions beside EB dots and in the control. Mast cells also aggregated and degranulated to release algogenic substances of 5-HT and HA in the EB dots. Our results indicates that the mechanism of EB extravasation in the skin of the acupoints are closely related to neurogenic inflammation induced by GMI and that the high expression of local allergic substances and nociceptive neuropeptides may be the underlying mechanism of acupoints sensitization.
Relationship between segmental distribution of EB dots and acupoints
Some of the cutaneous EB dots appeared in the exact locations of acupoints of BL20, BL21, DU6, BL17, RN12, and RN13. These acupoints are frequently used to treat gastrointestinal diseases in Chinese medicine [11, 12]. The EB dots appeared after GMI, and disappeared gradually during the natural self-recovery of the gastric mucosa, which indicated that the local neurochemical substances of the EB dots changed with the different degree of GMI. When the gastric mucosa was injured, most homosegmental acupoints were also “sensitized” and in an active state to reflect the disease, whereas, when the GMI subsided the function of these acupoints recovered to their latent state gradually. The distribution of the sensitized acupoints after GMI was restricted to the dermatomes of T9-11 in the same somite with gastry, which indicated that acupoints in the same somite of suffered viscera tended to be sensitized more than other acupints.
Neuronal pathways mediating referred pain in the skin of rats after GMI
Mast cells were activated by nociceptive neuropeptides and then aggregated, degranulated, and released algogenic substances in EB dots
Mast cells are effector cells in allergic and anaphylactic reactions. However, during inflammatory responses mast cells can also respond to stimuli such as neuropeptides of SP, CGRP, independent of FceRI . Tryptase is a neutral protease selectively concentrated in the secretory granules of mast cells (but not basophils), serving as a marker of mast-cell activation . In this study mast cells in the skin of sensitized acupoints were activated to respond to neuropeptide SP and CGRP. They gathered, degranulated, and released 5-HT and HA in EB Dots (Fig. 8). Because of their anatomic association with cutaneous nerves, mast cells and their released substances appear to play an important role in mediating neuronal antidromic responses in the skin, although the precise role of these cells in cutaneous inflammation remains to be determined .
Previous studies showed that mast cells participated in the mechanism of analgesia induced by acupuncture [24, 25], moxibustion , and laser acupuncture . Once mast cells are activated, the released mediators can be expected to activate sensory nerve fibers , and some of them, such as adenosine, have been suggested as a mediator of acupuncture-induced analgesia . Further research is needed to investigate the role of mast cells in the effect of acupuncturing at sensitized acupoints.
Revelation of paradoxes in acupoints research
Recently a number of well-designed clinical trials and systems reviews have reported that true acupuncture is superior to routine medication, but does not significantly outperform sham acupuncture. These findings are apparently at odds with traditional theories regarding acupuncture point specificity . On the other hand, evidence from laboratory shows that stimulation of different points on the body causes distinct responses in hemodynamic, fMRI, and central neural electrophysiological responses . These paradoxes in acupuncture research bring us a vital question. Is it reasonable to consider positions beside the acupoints as sham acupoints or nonacupoints?
This study sheds light on the paradox of acupoints. It indicates that the sensitized acupoints are limited to the related dermatomes and the location is not limited to a point because the area beside the EB point is also sensitized by increased expression of nociceptive substances. These results suggest that the areas beside EB dots are also activated, although less than the EB dots. Thus, sham acupoints beside the real acupoints are not an appropriate control.
HCl-induced GMI results in neurogenic plasma extravasation in the skin of acupoints limited to T9-11 dermatomere. In the extravasated dots there is a biochemical milieu of substances associated with neuroinflammation, which includes elevated expressions of SP and CGRP in nerve fibers, and elevated expressions of HA and 5-HT in activated mast cells. The extravasated dots are an indication of sensitized acupoints, and are considered as an active response to the disease. The present study gives, for the first time, evidence of kinetic process of acupoints sensitization and its underappreciated mechanism. Further research is needed in regard to the effect of acupuncturing at sensitized acupoints.
Gastric mucosal injury
Calcitonin gene-related peptide
We thank Dr. Nissi Wang and Wan-Xin Liu for word polishing.
This work was supported by grants from the National Natural Science Foundation of China (No. 81330087, 81273829), the national basic research program of China (973 program, No. 2011CB505201).
Availability of data and materials
The datasets during and/or analysed during the current study available from the corresponding author on reasonable request.
XHJ and BZ conceived and designed the experiments. WH, XYW, HS, BC, YSS performed the experiments. WH and WZB analyzed image and the data. XHJ, WH and XCY wrote the paper. All the authors approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The study was approved by our Ethics Committee of Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences.
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