| Objective To establish an neonatal rat necrotizing enterrocolitis model (necrotizingenterrocolitis, NEC) by using hypoxia and reoxygenation, cold exposure, artificalfeeding．To administrate chitosan oligosaccharide (Chitosan oligosaccharide, COS) asintervention, observer the protective effects of COS on intestinal mucosa of neonatal ratswith necrotizing enterocolitis, and to provide an experimental basis for the prevention andtreatment of NEC by administration of COS.Methods Selected5female pregnancing SD rats on the same day (The rats wereprovided by experimental animal center of Soochow University), then selected36newbornrats of the birth weight over5g after the natural childbirth. the average weight was about5.5±0.4g, either gender. They were randomly divided into model group (group A),chitosan oligosaccharide intervention group (group B) and normal control group (group C),each group including12. All the neonatal rats were fed with breast milk in2days afterbirth. Then Group A and group B were separated with their mothers, given artificialfeeding. The rats of group A were fed with simulated milk only (once every4hours), andwere given4℃cold exposure10minutes after hypoxic-reoxygen(100%nitrogen about90seconds,100%oxygen about3minutes)treatment (twice a day for3days); the rats ofgroup B were fed with COS and simulated milk (once every4hours), and were given4℃cold exposure10minutes after hypoxic-reoxygen(100%nitrogen about90seconds,100%oxygen about3minutes)treatment (twice a day for3days); the rats of group C were stillmaternal breast-fed, without any treatment. Weights of all the rats were weighed at thesame time every day. On the sixth day, all the neonatal rats were fasted for24hours. Thenall the rats were killed by cervical dislocation, Take ileum tissue about1.5cm to2cm forpathological examination, TUNEL detection and electronmicroscope observation. The remaining intestinal tissue homogenate, PAF and SIgA level were tested by ELISA.Results1. The growth and development in different groupGroup A and group B neonatal rats began to appear different degrees of activitydecreased, abdominal distension, diarrhea, weight loss during the second day, in group Aneonatal rats also appears twitches, even death; in group C rats were normal activity,sensitive reaction, gaining more weight, eating and defecation were normal. During theexperiment, in group A2cases were death at fourth day, while no death in group B andgroup C. Each group got weight increase, group A:0.96±0.23g, group B:1.99±0.56g,group C:4.27±0.28g. There was significantly difference between group A and group B ((P<0.05).2. Path morphological of the ileum tissue in each groupThe average histological score of each group(A、B、C)was as follows：2.92±0.93,2.00±0.64,0.38±0.31, group B compared with group A, the difference was statisticallysignificant (P <0.05). In group A the score on a scale of2to4, Specific performance isthat intestinal tissue isolated from the intestinal mucosa and (or) the lamina propriamoderate, or moderate edema from the intestinal submucosal and muscle layer to allintestinal villi disappear intestinal necrosis, with typical pathological changes of NEC; Inthe chitooligosaccharides intervention group, the score ranged from1to3, from mildintestinal mucosa and (or) the lamina propria of separation to the intestinal mucosa and (or)intrinsic layer re-degree separation, or from under the intestinal mucosa and muscle layerheavy degree of edema, local intestinal villidisappear off. In group C the score on a scaleof0to1，the structure of intestinal mucosal epithelial tissue is complete.3. Concentration of PAF in each groupConcentrations of PAF in group A、B and C were33.91±1.87μg/ml,25.21±1.76μg/ml,17.55±1.87μg/ml, Group A was significantly higher than group B, There was significantlydifference between group A and group B (P <0.05).Concentrations of SIgA in group A、B and C were24.75±1.83μg/ml、31.81±1.31μg/ml、36.33±1.58μg/ml. Group A was significantly lower than group B. The level ofgroup A was significantly lower than that of group B (P <0.05).4. Electron microscope observation: Group A showed that apoptotic body existed,suggesting that the advanced stage phase of apoptosis; Group G showed that condensed chromatin marinated around the nuclear envelope, nuclear pores expanded, suggesting theearly phase of apoptosis; Group C showed that its cell volume was mostly occupied by thenucleus, the structure was clear, nuclear membrane existed, suggesting the normal phase ofcell.5. The apoptosis rate of intestinal epithelial cells by TUNEL detection (%): group A44.17±4.37; group B27.33±5.21, group C9.00±2.86. the apoptosis rate of intestinalepithelial cells of Group B was significantly lower than that of group A. There wassignificantly difference between group A and group B (P<0.05).Conclusion1. The method of using hypoxic-reoxygen, cold exposure, artifical feeding couldestablish NEC model. The pathological changes of NEC model are more consistent withthe pathological features of neonatal NEC.2. Compared with the model group, the COS intervention group significantly reducedintestinal mucosal injury, show that COS has the protective effect on guts of in neonatalrats with NEC, which work by inhibiting the expression of the inflammatory cascade inPAF, improving intestinal secretion of SIgA, inhibiting of intestinal mucosal epithelial cellapoptosis, releasing intestinal tissue injury... |
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