| Objective:1 To compare the incidence of peripheral nerve lesions of Hartley guinea pigs induced by oral-infused Campylobacter jejuni of different sources.2 Establishment of guinea pigs model for peripheral nerve injury induced by oral-infused Campylobacter jejuni.Method:1 Select bacterial strains: Lulei strains and Lichang strains of Campylobacter jejuni isolated from stool samples of GBS patients, to compare which bacterial strains is better to construct animal modal; NCTC11168 strains isolated from stool samples of diarrhea patients, wether they will induce the damage of peripheral nerve of experimental animals; Lulei variation strains isolated from stool samples of mice by Liu Luji repeatedly lavage mice with Lulei strains, they caused a mice paralysis and eventually death, in this experiment to further observe the force of inducing peripheral neuropathy; Fowl strains isolated from stool samples of chicken, which C. jejuni as a natural host for avian, through this experiment to observe the force of inducing peripheral neuropathy; Xiao Tian was isolated from fecal samples of GBS patients who was hospitalized in the second hospital of Hebei medical university in 2013, to compare the virulence of C. jejuni with C. jejuni of GBS source of 20 years ago.2 Campylobacter jejuni culture, identification and bacterial suspension preparation: The frozen six bacterial strains resuscitated, were incubated under laboratory standard micro-aerobic conditions(42℃,10%CO2、5%O2、85%N2) for 24 hours and then were incubated to third generation. They were identified based on gram-staining and hippuric acid hydrolysis test. Then they were purified and suspended to concentration of 3 × 109 CFU/ml.3 Experimental animal groups: 56 Hartley guinea pigs were randomly divided into six experimental groups and a control group. According to the different oral-infused bacterial strains, the experimental groups were divided into Lu Lei group, Lu Lei variation group, Li Chang group, Xiao Tian group, Fowl group and NCTC11168 group, with 8 guinea pigs in each group.4 Experimental animal treatment: Guinea pigs were fasted for 12 hours, deprived of water for 3 hours before oral-infused bacterial strains. Lu Lei group, Lu Lei variation group, Li Chang group, Xiao Tian group, Fowl group and NCTC11168 group were respectively fed 1ml bacterial suspension of Lu Lei strains, Lu Lei variation strains, Li Chang strains, Xiao Tian strains, Fowl strains and NCTC11168 strains with a concentration of 3 × 109 CFU/ml per animal; the control group was given 1ml Brucella broth.5 Experimental animal observations and anatomy: Experimental animals were observed every day and weighed regularly. Standards of grading of gunuin pigs paralysis reference that of EAN modal of Hartley gunuin pigs: According to paralysis degree devided into 0-5 grades, 0 grade: nomal; 1grade: toes of two hind legs slightly drag; 2 grade: toes of two hind legs obviously drag, but others are normal; 3 grade: two hind legs obviously drag, usually two hind legs incline to one side; 4 grade: two hind legs completely paralyse; 5 grade: quadriplegia, even respiratory depression.Two guinea pigs in each group fed with bacterial strains for 3 weeks were dissected in the fourth week since the first treatment, 2 of every group per week were dissected, respectively in the fifth, the sixth and the seventh week. The sciatic nerves were dissected and conserved in 4% paraformaldehyde. Then the samples were observed by osmium tetroxide staining. We also observed whether the gastrointestinal tract of experimental animal suffered from edema, hemorrhage and necrosis etc.6 Statistical analysis:100 teasing fibers were examinated and took photos, then computing the incidence of peripheral nerve lesions(The incidence of peripheral nerve lesions=damage fibers/100×100%). Compare incidence of pathological fibers in every group by SPSS 13.0, whether there are different. To examinate whether there were statistically significant difference among the weight of seven group by repeated measurement data.Results: 1 Clinical observation of the experimental animal: 1.1 Reference guinea paralysis score: the scores of all groups were 0. Two guinea pigs of Fowl goup occured small pieces of hair thinning; three guinea pigs of NCTC11168 group suffered from hair removal, one died from intestinal prolapsed; two guinea pigs of Li Chang group suffered from hair removal, one turn over itself poorly, which recovered after two days; two guinea pigs of Lu Lei variation group suffered from hair removal. Others did not occur diarrhea, paralysis, hair removal and so on during the period of observation. 1.2 Statistical analysis of weight change in every group: There were not statistically significant difference among the weight of seven group by repeated measurement data. 1.3 The general observation of the gastrointestinal tract of experimental animal: NCTC11168 group no.6 guinea pig suffered from intestinal prolapsed in the 27 th after the first oral-infused C. jejuni that is the 4th after the last one. The others is normal. 2 Peripheral nerves manifestation of osmium tetroxide staining:The control group: These fibers demonstrate that the myelins and nodes of Ranvier are normal.The experimental group: These fibers demonstrate that paranodal reg-ion of myelin sheath shrink into dentation or the “Great Wall”, demyelin-ation gathered into sheet or oval shape, nerve fiber is uneven thickness, nodes of Ranvier lengthen, nerve fibers have a wormhole-like coloboma and the whole nerve fiber shrink etc. 3 Incidence of peripheral nerve lesions by the statistical analysis:We use SPSS 13.0 to process the data. Chi-square test with bilateral testing were used to analysis the incidence of nerve fiber lesions and α= 0.05 was set as the test level. The results of morphologic examination on peripheral nerves showed that for teased-fiber study, incidence of nerve fibers lesions from different groups is statistically significant different. Any two groups were further compared by partition method of chi-square test and correcting the test level. The results showed that any experimental group was statistically significant different from the control group. The incidence of all groups range from high to low: Lulei group(32%)>Fowl group(21.88%)>Lulei variation group(20.88%)>Li Chang group(19.75%)>Xiaotian group(17.38%)>NCTC11168 group(15.63%)>the control group(4.38%); Lu Lei group was statistically significant different from other groups, and its incidence was highest than other groups; NCTC11168 group was statistically significant different from Lu Lei group and Fowl group, and its incidence was lower than the two group; No statistically significant difference was found among NCTC11168 group, Lu Lei variation group, Li Chang group and Xiaotian group.Conclusions:1 In this experiment, Campylobacter jejuni Lulei strain, Li Chang strain,and Xiaotian strain which derived from GBS patients, Fowl strain derived from chicken,NCTC11168 derived from diarrhea patients,and Lulei variation strain from different growth states can induce each guinea pig to suffer from peripheral neuropathy, but each guinea pig has different incidence of peripheral neuropathy. The incidence of peripheral nerve lesions induced by Campylobacter jejuni derived from GBS pati-ents characterized by AMAN was the highest, and that of NCTC11168 was the lowest.2 C. jejuni-NCTC11168 which had not been payed attention to can induce peripheral neuropathy and derived from diarrhea patients also found guinea pig to suffer from peripheral neuropathy. |
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