| Objective1 The study provided evidence that C.jejuni infection in the guinea pigs can provide an experimental animal model of peripheral neuropathy. The original C.jejuni strain was isolated from the stool of a patient with AMAN(Acute Motor Axonal Neuropathy,). 2 we studied the pathological changes in C.jejuni-induced guinea pigs model for peripheral neuropathy.C.jejuni and placebo were fed to each guinea pigs.This study may provide more experimental data to establish perfect animal model for peripheral neuropathy associated C.jejuni.Methods1 Guinea pigs and infection of C.jejuni Four weeks old guinea pigs(white feathered,Hartely domesticus,N = 50) included in the study were purchased from Animal House of Hebei Medical University.Half were males and half females.Guinea pigs were provided free access to food and water and kept in separatecages at animal care facility of the institute.Guinea pigs were randomly assigned to experimental group and control group.The guinea pigs will be killed at 1st,2nd,3rd,4th and 5th weeks respectively when the C.jejuni feeding terminated,so experimental group was divided into five subgroups(6guinea pigs/group) according to the anatomy time:Group E1、E2、E3、E4、E5.Experimental group was fed with 1ml of 3 × 109CFUs/ml C.jejuni suspension.Control group was also further divided into two subgroups(10 guinea pigs/group): Group A: the guinea pigs were fed with Brucella broth,Group B: completely blank control. The original C.jejuni strain was isolated from the stool of a patient with AMAN and was typed as Penner 0:19 by the USPHS Centers for Disease Control(Atlanta,GA,USA).C.jejuni was incubated in microaerobic condition(42℃,5% O2,10% CO2,85% N2.) for 24 h and cultured on Colombia blood agar plate to 3th generation. Then C.jejuni was identified by Gram staining,hippurate hydrolysis test and PCR.We purificated the bacteria and prepared bacterial suspension with a concentration of 3×109cfu/ml. Foods were deprivated for 15 hours and water was deprivated for 4 hours before C.jejuni feeding.Guinea pigs of Control A group were orally fed Brucella broth and experimental group guinea pigs were orally fed 1ml C.jejuni suspension for 3 weeks,3 days a week,respectively. All procedures were in accordance with the guidelines of the China Council on Animal Care,the study was approved by the institute ethics committee. 2 Observation,Killing and tissue processing We observed Guinea pigs’ clinical symptoms every day and weighed guina pigs regularly.(Scoring standard reference the EAN model of the Hartley guinea pigs). Such as mental status,fur situation,symptoms of the gastrointestinal tract were observed.6 animals from each experimental group(E1,E2,E3,E4,E5) were killed respectively at 1st,2nd,3rd,4th and 5th weeks when the C.jejuni feeding terminated,2 animals from A group and 2 animals from B group were killed at the same moment.All the animals were euthanized and fixed by paraformaldehyde. The sciatic nerve samples were fixed in 4% paraformaldehyde solution at 4 ℃,buffered at p H 7.4 for 24 h.Sciatic nerves were observed by light microscopy after osmium tetroxide staining. And χ2 test were conducted to compare the lesion rate of peripheral nerves in Hartley guinea pig at different time,and to observe the changing process of neuropathology.Results1 Animal observation 1.1 In the experimental groups,an total of 6 guinea pigs became paralysed. Paralysis were observed in 4 guinea pigs immediately and also in 2 guinea pigs at the fourth week after intervention.According to the clinical symptom score standard,paralysis guinea pigs were graded 4-5,showing activity weakened,ears and lips cyanosis,shortness of breath,body tremblingslightly,dirty belly fur and poor bipedal action. In other expermental animals,slight alopeciaonly or no abnormal physical activity was observed,graded 0(Fig.5). 1.2 The body weight of experimental guinea pigs increased slower than control groups.But the weight was no statistically significant difference in experimental group and control group guinea pigs,F=4.925,P>0.05(Fig.6). 2 Peripheral nerve pathology The nerve fiber lesions rates of guinea pigs in experimental groups and control groups were different.As shown in(Table 2). Sciatic nerve fibers of guinea pigs in the control group also showed shrinkage and inhomogeneous,but we thought it was an nomal pathological apoptosis state(Fig.7).All guinea pigs in experimental groups had sciatic nerve lesions,pathology ranged from atypical to severe wallerian-like degeneration.The nerve change identified was lengthening of the nodes of Ranvier,often associated with distortion of the paranodal myelin and the myelin sheath attachment sites;the thickness of partial sciatic nerve fibers was inhomogeneous;the myelins were segmentally disappear and ruptured,and the outermost myelin lamellae at the paranode degenerated,just like the “Great Wall”(Fig.8). 3 Statistical analysis All the data were processed by SPSS 13.0.Chi-square test with bilateral significant testing were used to analysis the rates of nerve fiber lesions and α= 0.05 was set as the test level. We consider that the nerve fiber lesions rates of Hartley guinea pigs in different groups are not all equall( P<0.05). Further comparison among different groups used the partitions of χ2 method and α= 0.05 was set as the test level. The rates of nerve fiber lesion in paralyzed guinea pigs was 42.0%.the rates of experimental group E1,E2,E3,E4,E5 were 15.3%,18.0%,29.0%,39.0%,38.7% respectively(Table 2). The rate of nerve fiber lesions was statistically significant difference in experimental group and control group guinea pigs anatomed at the sameperiod,P<0.05,showing that the rate in the experimental group is higher than control group(Table 3). The nerve fiber lesions showed no statistically significant difference in Brinell broth group and control group,P>0.05(Table 3),showing that Brinell broth have no impact on nerve fibers. The rate of nerve fiber lesions was no statistically significant difference in Brinell broth group and control group guinea pigs anatomed at different time,P> 0.05(Table 4),showing that the time had no effect on the nerve fiber lesions. The rate of nerve fiber lesions was statistically significant in experimental group guinea pig anatomed at different time,P<0.05(Table 4),showing the rates are not all equal. For further comparison,we use χ2 segmentation method to set the test level as α ’= 0.0033.The rate of nerve fiber lesions is higher in group T and group E4 than other expermental groups,P<0.0033(Table 5).The rate showed no differences between E4 and paralyzed guinea pigs by the Chi-square Segmentation.and there is a certain correlation between time and nerve fiber lesions by spss13.0 curve fitting methods(Table 6).Conclusions1 The C.jejuni strain isolated from a patient with AMAN can lead to peripheral nerve injury in 4-week-old guinea pig by gastrointestinal infections. Paralysis were observed in some guinea pigs. 2 The nerve fibers lesions rate is different in 4-weeks-old guinea anatomed at different time after C.jejuni infection,the rate in group T and group E4 was higher than in other experment guinea pigs. |
PDF Full Text Request