| Objective: In this study,a method,using spatial autocorrelation and multivariate statistics,that extracts feature of distribution of Evans Blue(EB)exudation points in rat dorsal skin following acute gastric mucosal injury(AGMI)was presented.And thus we explore the correlation between the mathematical features of the body surface of acupoints and the pathological changes of zang-fu organs.Method: We first obtained experimental research conditions through pre-experiment.Then,in Experiment I,fifty-six healthy adult Wistar SPF rats,weighing 185 to 195 g 24h after fasting,were randomly divided into 7 groups(n?=?8 per group): normal group,saline group,0.40 M hydrochloride group(0.40M),0.45 M hydrochloride group(0.45M),0.50 M hydrochloride group(0,50M),0.55 M hydrochloride group(0.55M),0.60 M hydrochloride group(0.60M).The rat model of AGMI was treated with hydrochloric acid(HCL)through gastric gavage(1.5ml).Rats in saline group was intragastrically injected with an equal volume of saline.And the rats in normal group only performed grasping movement.Two rats from each group were randomly selected after 13 h modeling,and examined by histopathology of stomach specimens.Rats were anesthetized at 4h post-modeling,and injected with EB in the tail vein.And then,the rats were depilated and fixed in board and photographed at the same height at different time points at 5,7,9,11,and 13 h post modeling,respectively.We constructed a coordinate system on photos using the longitude and latitude grid counting method to record the number of EB exudation points and that position.Multivariate statistical analysis techniques including cluster analysis,factor analysis,correlation analysis and contour analysis were used to process the data.In experiment II,forty healthy adult Wistar SPF rats,weighing 185 to 195 g 24h after fasting,were randomly divided into five groups: blank group(BG),model group(MG),electroacupuncture BL-21 group(EA-BL21),electroacupuncture features area group(EA-FA),Gua-sha features area group(GS-FA).The rats were anesthetized before modeling,and depilated.Then,longitude and latitude grid were laid on the of rats dorsum in EA-FA and GS-FA groups.According the result of experiment I,the rat model of AGMI was treated with0.60 M HCL through gastric gavage(1.5ml).The rats in blank group were intragastrically injected with an equal volume of saline.Subsequently the rats were intervention at 13 hours post-modeling They were covered head by black cloths.The intervention methods are as following: the rats in blank and model groups only performed grasping movement.The rats in EA-BL21 group were treated with electroacupuncture at the bilateral Weishu(BL21)acupoints.In the EA-FA group,acupuncture was be performed at the midpoint of each grid in the "feature area",and the first and third needles were used with the EA.Needles were inserted about 10 mm deep and stimulated electrically at a frequency of 2 Hz continuous wave to permit the local muscle to shrink slightly.The duration of each stimulus was 30 minutes.In the GS-FA group,a small amount of vaseline was used on the skin of "feature area".The Gua-sha plate was oriented at approximately 45 degrees to the horizontal.The rat without struggling was considered as indicators for the stimulus intensity.The frequency of the scraping was set at 2 times/second and the number of that was 120 times.All procedures were performed once a day for 7 consecutive days by the same researcher.At the end of intervention,after fasting for 24 h,the rats were anaesthetized and stomachs samples were collected.Enzyme-linked immunosorbent assays(ELISA)were used for measurement of TNF-α,IL-1β,IL-6 and IL-8 concentrations in stomach homogenate.And for gastric histology,the longitudinal specimens were fixed with 4% paraformaldehyde,embedded in paraffin,and stained with hematoxylin and eosin(HE).Result: 1.In experiment I,histological examination of gastric tissue demonstrated that the gastric mucosal structures of normal and saline groups were intact with no obvious inflammatory cell infiltration.Mucosal shedding necrosis,edema,hyperemia,and inflammatory cell infiltration were observed in the gastric mucosa after HCL-gavaged.There was a positive relationship between injury degree and HCL concentration.2.The results of cluster and correlation analysis showed that IL12 was obtained via analyzing the data of EB exudation points.And the trend of temporal variation was negatively correlated with characteristics of exudation of 0.40 M concentration,that positive correlated with characteristics of exudation of 0.50 M concentration.The trends of concentration variation were negatively correlated with characteristics of exudation of 5,7 and 9 h after modeling.3.The results of factor and correlation analysis showed that “feature areas” were obtained via analyzing the data of EB exudation points,which characteristics of exudation were correlated with the trend of temporal variation,including 1,2 and 11 feature areas with stronger exudation tendency.4.“11 feature area”,“11’ feature area”;temporal variation profiling interactive analysis demonstrated that no statistically significant differences were observed between the two groups both parallel and coinciding profile analysis(P>0.05).It indicated that the profile of“11 feature area” was paralleled and coinciding with that of “11’ feature area”.That was,the trend and degree of exudation were approximately the same.5.In experiment II,histological examination of gastric tissue demonstrated that the gastric mucosal structures of blank group were intact with no obvious inflammatory cell infiltration.Mucosal damage,hyperemia,and a small amount of inflammatory cell infiltration were observed in the gastric mucosa in model group.Gastric mucosal epithelial structure of rats basically returned to normal,the arrangement of glands tended to be orderly,the structure was slightly loose,and no obvious inflammatory cell infiltration and congestion were observed in EA-BL21,EA-FA and GS-FA groups.6.TNF-α,IL-1β,IL-6 and IL-8 expression levels in the gastric tissue were significantly increased in model group,compared with blank group(P< 0.001),that were significantly decreased in EA-BL21,EA-FA and GS-FA groups compared with model group(P<0.01,P<0.001),and IL-6 expression levels in the gastric tissue were significantly decreased in EA-FA and GS-FA groups compared with EA-BL21 group(P<0.01,P<0.001).Conclusion: 1.It is feasible to use spatial autocorrelation and multivariate statistics to extract and analyze the feature of distribution of EB exudation points in rat dorsal skin following AGMI.Meanwhile this study was carried out at different points in time and different concentrations.Therefore,we obtained the body surface position and exudation trend of "feature grid/region".2.EA-BL21,EA-FA and GS-FA groups markedly reduced the TNF-α,IL-1β,IL-6 and IL-8 expression levels in the gastric tissue.IL-6 expression levels in the gastric tissue were significantly decreased in EA-FA groups compared with EA-BL21 group. |
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