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Peripheral Neuropathy in Different Week Old Hartley Guinea Pig by Feeding Campylobacter Jejuni

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Tutor: DongMei;LiZhenZhong
School: Hebei Medical University
Course: Neurology
Keywords: Animal model,Campylobacter jejuni,Subclinical peripheralneuropathy,Guillain-Barr
CLC: R745
Type: Master's thesis
Year:  2014
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Objective:1To develop a peripheral neuropathy animal model in different week oldHartley guinea pig by feeding Campylobacter jejuni.2Observe the peripheral nerve pathology in different week old Hartleyguinea pig by feeding Campylobacter jejuni and find out the best age todevelop a robust animal model.Methods:1Select strains: The Campylobacter jejuni used in this experiment wasisolated from a patient with acute motor axonal neuropathy(AMAN), whosename is lulei.2Preparation of Bacterial suspension: The frozen strain were thawed,passaged and purified, then suspended to a final concentration of3×108CFUs/ml. Identification of Campylobacter jejuni use the gram stain and hippuricacid hydrolysis test.3Experimental groups: A total of243~5weeks old healthy guinea pigswere divided into experimental group (n=18) and control group (n=6). Theexperimental group was divided into3-week-old group (group A),4-week-old group (group B) and5-week-old group (group C) with6in each.The control group was divided into Brucella broth group (group D) and Blankgroup (group E) with3in each.4Intragastric administration: Kept the Hartley guinea pigs from the foodfor12hours and the water for4hours before intragastric administration. EachHartley guinea pig of group A, B, C was orally fed with2ml of3×108CFUs/ml Campylobacter jejuni bacterial suspension3continuous days a week for4weeks, The control group D was fed with same amount of Brucella broth.Hartley guinea pigs were given food and water immediately after fed with Campylobacter jejuni bacterial suspension or Brucella broth.5Observation of the experimental animal: Since the intragastricadministration their clinical symptoms (Scoring standard reference the EANmodel of the Hartley guinea pigs), mental status, fur situation, symptoms ofthe gastrointestinal tract were observed.6Dissection of the experimental animal: After one week of the lastIntragastric administration with Campylobacter jejuni bacterial suspension orBrucella broth to Hartley guinea pigs, take on both sides of the sciatic nerve.7Neuropathological examination of the sciatic nerve: After secondarymaterials, the left side of the sciatic nerve did transmission electronmicroscopy observation of nerve pathological changes, the right sciatic nervedid osmium tetroxide staining. Each Hartley guinea pig was randomlyseparated100nerve monofilaments. Single blind method was used in the lightmicroscope to observe the change of neuropathology and compare the lesionrate of peripheral nerves in Hartley guinea pig of different ages.Results:1Clinical observation of the experimental animalAfter fed campylobacter jejuni on Hartley guinea pig, according toHartley guinea pig EAN model standard, gives them scores based on theirclinical manifestation, Group A, B, C, D, E all score0and had no clinicalmanifestation. But after20days of intragastric administration, NO.1, NO.2,NO.5of4-week-old and NO.1of3-week-old Hartley guinea pigs’belly aredirty, the others are clean. All Hartley guinea pigs did not appear listlessness,diarrhea, etc.2Peripheral neuropathyOsmium tetroxide staining results: sciatic nerve of the Hartley guineapigs in experimental group show nerve monofilament uneven thickness,axonal swelling and myelin segmental retraction,"great wall" changes.Control group Hartley guinea pigs sciatic nerve show nerve monofilamentsmooth edges and uniform thickness.Transmission electron microscope: The result is consistent with the light microscope result. The experimental group Hartley guinea pigs sciatic nervepathological changes was variable severity of axonal degeneration. Mildaxonal degeneration characterized by microfilament, microtubule sparse andstructural disorder, mitochondria cristae and membrane fusion, mitochondriaempty sample. Myelin is complete. Demyelination and myelin stratification.Severe axonal degeneration, in which axons disappear, with subsequentmyelin breakdown and formation of amorphous globules. The control groupHartley guinea pigs sciatic nerve pathological was the normal microtubles,microfilament and mitochondria structure of axoplasmic contents, the myelinwas onion-like arrangement.3Statistical analysis of osmium tetroxide staining resultsOsmium tetroxide staining showed that the lesion rate of peripheralnerves in Hartley guinea pig of different ages is different. The results wereprocessed by SPSS13.0, chi-square test were used to analysis the number oflesion monofilaments in Hartley guinea pig of different ages. The P value of <0.05(χ2=249.41)determined that we reject the null hypothesis and accept thealternative hypothesis and the lesion rate of peripheral nerves in Hartleyguinea pig of different ages was different. The result would be consideredstatistically significant. Any two groups were further compared by chi-squaretest partition method and α′=0.005as significant level (α=0.05) depending onthe study. Pairwise comparison showed4-week-old guinea pig nervemonofilament lesion rate was significantly higher than3-week-old group,5-week-old group, brucella broth group and blank group. The rate of3-week-old group,4-week-old group,5-week-old group were significantlyhigher than brucella broth group and blank group. There are no statisticallysignificant difference between3-week-old and5-week-old group, and betweenbrucella broth group and blank group.Conclusion:1Hartley guinea pig naturally infected with Campylobacter jejuni cancause14.00%to38.00%of the sciatic nerve injury monofilament, Do notappear listlessness, diarrhea, limb activity obstacle, etc. Therefore, Hartley guinea pig naturally infected with Campylobacter jejuni can lead to subclinicalperipheral neuropathy.2Osmium tetroxide staining showed the lesion rate of peripheral nerve inHartley guinea of different weeks is different and the4-week-old guinea pignerve lesion rate is the highest through chi-square test. This will lay thefoundation of animal model for the follow-up study that the guinea pig aged4weeks naturally infected with Campylobacter jejuni cause Guillain-Barresyndrome.3The peripheral neuropathy in Hartley guinea pig naturally infected withCampylobacter jejuni could be the early changes of pathology in Guillain-Barre syndrome based on osmium tetroxide staining and transmission electronmicroscope. During the follow-up study we need to determine anti-GM1andanti-GD1b, extend the observation time after intragastric administration, lookfor ways to observe mild weakness on Hartley guinea pig and build GBSanimal model of Hartley guinea pig further.
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