The Damage Of Peripheral Nerve In Hartley Guinea Pigs Has Arised Along With The Increase Of Feeding Frequency Of Campylobacter Jejuni

Objective: 1 Whether the frequency of gastric lavage will affect the peripheral nerve injury of Hartley guinea pigs under the condition of pertussis adjuvant. 2 The present study is designed to provide the basis for the further development of peripheral neuropathy models of Hartley guinea pigs caused by Campylobacter jejuni through natural infection and the development of Vaccine.Methods: 1 Strains: We selected Lulei strain from the library of C.jejuni strains as experimental strains. 2 Preparation of Bacterial suspension: Recovered the cryopreservated Campylobacter jejuni Lulei strains and subcultured it, then we used the morphological, biochemical tests to identificate it, finally prepare the purified Campylobacter bacteria to broth suspension at a concentration of 3×108cfu/ml. 3 The groups of Animal experiment : After 1 weeks of adaptated feeding, we screened 28 healthy guinea pigs from 32, which are the SPF level of 4 week old, according to weight, weight growth rate and the difficulty of stomach. These animals were randomly divided into 2 groups and 1 control group, further, the experimental groups were divided into A group and B group according to different feeding frequency, 12 guinea pigs in each experimental group and 4 guinea pigs in the control group. Based on the anatomy of different time(the sixth day,the twelfth day, the eighteenth day,the twenty-fourth day after the last feeding irrigation), the experimental groups and control group were randomly divided into 4 subgroups, name A1, A2, A3, A4; B1, B2, B3, B4; C1, C2, C3, C4. There were 3 guinea pigs in each experimental subgroup, and 1 guinea pig in each control subgroup. 4 Plan of poison attack: On the first day and seventh day of stomach, Hartley guinea pigs of each group were intraperitoneal injected 2 times of pertussis toxin 0.5ml at a concentration of 1g/ml. The animals in group A were gavaged every 2 days to a total of 10 times, animals in group B were gavaged every 3 days to a total of 7 times, the control group C was only intraperitoneal injection of pertussis toxin. Before fed, Hartley guinea pigs were fasted 12 hours of food and 3 hours of water deprivation. Each Hartley guinea pigs of group A or group B were fed with 3×108cfu/ml Campylobacter bacteria suspension 2ml. Hartley guinea pigs were immediately given food and water after gavaging. 5 Observe the experimental animal: Since adaptive period, we observed the activity, daily mental state, fur color, sound and diarrhea of Hartley guinea pigs every day and regularly weighed them. And compared the difference between body weight of guinea pigs in differdent groups through analysis of variance of repeated measurement. 6 Dissect the experimental animal: On the sixth day after the last feeding, we dissected group A1, group B1 and group C1; and on the twelfth day, dissected group A2, group B2 and group C2; on the eighteenth day, dissected group A3, group B3 and group C3; on the twenty-fourth day, dissected group A4, group B4 and group C4. After anesthetized the guinea pigs with 4% chloral hydrate(10ug/g), we perfused them with 4% paraformaldehyde, and dissected the bilateral sciatic nerve proximal, and then preserved them in 4% paraformaldehyde to practice osmium acid staining, hematoxylin and eosin and Luxol-fast-blue silver staining. 7 Pathological observation and statistics of the sciatic nerve: After two times of anatomy and paraffin embedded, the right sciatic nerve of each guinea pig executed HE staining and Luxol-fast-blue silver staining to observe the pathological changes. The 100 nerve filaments were separated from the left sciatic nerve of each guinea pig which were executed osmic acid staining. We choosed single blind method to observe them under the light microscope, and calculated the number of the diseased nerve filaments and calculation of the incidence rate(peripheral neuropathy monofilament rate = lesions filament count /100*100%). All the data were analyzed by statistical software SPSS13.0. Chi square test was used to compare the rate of peripheral neuropathy monofilament between different groups and subgroups.Results: 1 Observe the experimental animal 1.1 Clinical symptoms After oral gavaged with Campylobacter jejuni, the Hartley guinea pigs scored 0 stage according to the standard for evaluation of clinical symptoms(reference standard for evaluation of Hartley guinea pig EAN model). The second day after the fourth time of irrigation stomach(that is, the eighth day after the first time of irrigation stomach), No. 25, No. 30, No. 32 and No. 27 guinea pigs in experimental group A appeared hypokinesia and struggle with weak when catching them, No. 27 guinea pig also appeared lower volume of sound. All the guinea pigs were returned to normal in 2 days. The sixth day after the last irrigation stomach(that is, the twenty-second day after the first time of intragastric administration), No.32 guinea pig appeared hip depilation, the syptom began to improve in the following 9 days and did not fully restore until the autopsy. 1.2 Body weight Through the analysis of repeated measures ANOVA, there is no significant difference about body weight of guinea pigs among the control group and the experimental groups(P > 0.05). 2 Peripheral neuropathy 2.1 Osmium acid staining Sciatic nerve monofilaments of the Hartley guinea pigs in experimental group showed uneven thickness, in the inside of myelin protrusions,myelin segmental retraction and deletion; oval shaped myelin material deposition and wormhole like defect often seen at the node of Ranvier;myelin lateral serrations, “the Great Wall " changes.The nerve fiber of the No. 52 guinea pig of experimental group B appeared obviously " Wallerian like” degeneration. Sciatic nerve monofilaments of the Hartley guinea pigs in control group showed smooth edges and uniform thickness. 2.2 Hematoxylin and eosin staining The experimental group showed myelin loss, axonal fracture and constriction. The control group showed axonal smooth, uniform thickness. 2.3 Luxol-fast-blue silver staining Silver staining: The experiment group showed axonal fracture and constriction.Luxol-fast-blue silver staining: The experimental group showed myelin loss, axonal fracture and constriction. 3 Statistical analysis of peripheral neuropathy monofilament rate All the guinea pigs in the experimental groups existed peripheral nerve injury.The rate of nerve monofilament lesion in the control group was 4.75%, the rate of nerve filament lesions in the experimental group A and group B were 45.58% and 32.17% seprately. The rate of neural filament lesions of the No.17 guinea pig in group A and the No.57 guinea pig in group B were 34%, 45%. According to the statistics they were the outliers. Excluding outliers, the rate of nerve filaments lesions in the control group was 4.75%, in the experimental group A and group B respectively were 46.64%, 31.00%.Using SPSS 13.0 statistical software for statistical analysis: 1, When didn't remove the outliers, chi-square test was used to analysis the rate of lesion monofilaments of different groups. The result( P < 0.05,?2=226.055) showed that the lesion rate of peripheral nerves in Hartley guinea pig of different groups was different. Further comparation between any two groups were used chi-square test partition method and ?'=0.0167 was seen as significant level(?=0.05). Pairwise comparison showed guinea pigs nerve monofilament lesion rate of group A was significantly higher than that of group B and group C. Goup B was significantly higher than group C, with statistical significance. 2,When remove the outliers, the result( P < 0.05, ?2=236.430) suggested that the lesion rate of peripheral nerves in Hartley guinea pig of different groups was different. Pairwise comparison showed the rate of nerve monofilament lesion of group A was significantly higher than that of group B and group C, goup B was significantly higher than group C. So, whether elimination of outliers, the rate of nerve monofilament lesions was consistent. According to different anatomical points, the rate of nerve filaments lesions of group A1, A2, A3, A4 respectively was 43.33%, 45.67%, 49.00%, 44.33%. The rate of nerve filaments lesions of group B1, B2, B3, B4 respectively was 30.00%,35.33%,29.00%,34.33%.The data were analyzed by chi-square test, bilateral inspection, take ? =0.05 level. 1, compared the four subgroups of group A, the result(P >0.05, ?2=2.214) showed that there was no significant differences. 2, compared the four subgroups of group B, the result(P >0.05, ?2=4.048) showed that there was no significant difference. From the first day to the twenty-fourth day after the last poison attack, the injury of peripheral nerve filament maintains a relatively constant level. There is no obvious trend of aggravation and mitigation.Conclusion: 1 Lulei strain from the library of C.jejuni strains can lead to peripheral nerve injury of all guinea pigs via oral gavage. 2 Along with the increase of feeding frequency of Campylobacter jejuni, the damage of peripheral nerve in Hartley guinea pigs has arised under the condition of pertussis adjuvant. 3 The self-recovery time of peripheral nerve is more than 24 days.