| Objective:1 Oberseve whether Campylobacter jejuni under poor condition infected Hartley guinea pigs through digestive tract causes peripheral nerve lesions.2 Compare the peripheral nerve lesions rate of Hartley guinea pigs induced by gastrointestinal infections of Campylobacter jejuni under different cultivation conditions.3 Establish and complete a natural animal model of peripheral nerve lesions caused by Campylobacter jejuni infected Hartley guinea pigs through digestive tract.Methods:1 Bacterial Strain:We choosed strain of C.jejuni from strains library named Lulei.2 Anabiosis,Culture,identification of Campylobacter jejuni and The preparation of bacterium suspension: The anabiosis of Lulei strain under ambient temperature?25??.The strain was cultured in 42? microaerophilic?5%O2,10%CO2,85%N2?incubator for 24 hours and passaged to third generation in medium.After confirmed by morphological and biochemical and PCR tests,remove half of the mediums to 42? atmosphere?21%O2,1%CO2,78% N2?incubator.The bacterial strains were collected and purified,then turn into two different concentration of 3×108CFU/ml bacterium suspension eventually and prepare for intragastric administration to guinea pigs.3 Experimental animal groups: 32 SPF Hartley guinea pigs of 4 weeks old were randomly divided into 2 experimental groups and a blank control group.According to the different cultivation conditions,the experimental groups were divided into microaerophilic group and aerobiotic group.Each experimental group contain 12 guinea pigs,blank control group have 8 guinea pigs.4 Treatment: Microaerophilic group each guinea pig was given by gavage 2 ml bacterium suspension from microaerophilic condition?turbidity reach 3×108CFU/ml?twice a day for 7 times totally.Aerobiotic group each guinea pig was given by gavage 2 ml bacterium suspension from atmosphere condition?turbidity reach 3×108CFU/ml?twice a day for 7 times totally.Control group received no treatment.Before intragastric administration every guinea pig fast solids for 12 hours and liquids for 3 hours.5.Animal Observation: The weight of guinea pigs was measured regularly.The general condition?movement,food taking,spirit?and clinical symptoms?diarrhea,paralysis?was observed every day after intragastric administration.Standards of grading of guinea pigs paralysis reference the EAN modal of Hartley guinea pigs6 Animal dissection: After the last intragastric administration,we dissected 8 guinea pigs every 5 day,3 from each experimental group and the other 2 from blank control group.The sciatic nerves were dissected and conserved in 4% paraformaldehyde.Then we stained sciatic nerve by osmium acid and hematoxylin with eosin after second processing.7 Nerve pathological change and statistical analysis: After stained by hematoxylin and eosin,we observed sciatic never under light microscope.we separate 100 nerve fibers randomly in each guinea pig under the stereomicroscope after osmium acid.These nerve fibers were observed and evaluated pathologic degree under the microscope.Pathological nerves were recorded,photographed and calculated in rate of lesions.All the data were processed by SPSS 13.0 statistical package.Chi-square test with bilateral significant testing were used to analysis the rates of nerve fiber lesionsResults: 1 Observation: 1.1 Clinical symptoms: According to EAN model paralysis score,each group get o score.6 guinea pigs in control group developed obvious hair removal.Others are not observed hair removal,diarrhea,paralysis etc.1.2 Body weight: The body weight of both experimental group and blank control group floated up and down.There were no statistically significant difference among the weight of each group by repeated measurement method.2 Sciatic nerve staining 2.1 Osmium tetroxide staining:The control group: These nerve fibers run smoothly and the myelins and structural integrity of nodes of ranvie,less crenated myelin.The experimental group: Every guines pig was observed nerve lesions.These can be seen that uneven nerve fiber,structural defect of nodes of Ranvie,crenated myelin,flaky and oval deposits,slight wallerian-like degeneration.2.2 HE stainingThe experimental group: These nerve fibers were observed demyelination and axonotmesis.The control group: These nerve fibers run smoothly and also the myelins,were observed structural integrity of axon.3 Statistical analysis:All the data were processed by SPSS 13.0 statistical package.Chi-square test with bilateral significant testing were used to analysis the rates of nerve fiber lesions and ?= 0.05 was set as the test level.It is concluded that the P value<0.05,then we consider that the nerve fiber lesions rate of Hartley guinea pigs in three groups are not all equal?P<0.05?.Any two groups were further compared by partition method of chi-square test and correcting the test level??=0.05?.The results showed that microaerophilic experimental group was statistically significant different from control group.And aerobic experimental group was statistically significant different from control group.Each experimental group were compared with control group and correcting the test level??=0.0125?.The results showed that two experimental group was statistically significant different.The lesions rate from high to low: M group>N group>B group.The results showed that three groups were statistically significant different.Conclusions:1 Lulei strain in strains library exposed to aerobic conditions?42?,21%O2,1%CO2,78% N2?still can lead to peripheral nerve injury in guinea pig by gastrointestinal infections.2 Campylobacter jejuni under microaerophilic?42?,5%O2,10%CO2,85%N2?and aerobic condition?42?,21%O2,1%CO2,78% N2?both can lead to peripheral nerve injury in guinea pig,and every guinea pig in experimental group had peripheral neuropathy on biopsy.3 The rate of peripheral nerve injury of guinea pig in microaerophilic group was higher than aerobic group. |
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