Gastrointestinal Infection Of Kunming Mice By Guillain-barre Syndrome Related Campylobacter Jejuni And The Classification Of Ischemic Stroke In The Very Elderly People

PART I The separation of guillain barre syndrome related Campylobacter jejuni in the feces and gastrointestinal tissues of Kunming miceObject:Through separating of campylobacer jejuni(C.jejuni) in gastrointestinal(GI) tissues and feces of Kunming(KM) mice gavaged with guillain Barre syndrom(GBS) related C.jejuni,to study the influence of different inoculating concentration, inoculating frequency, the age of mice,and the usage of pertussis toxin on the existent interval of C.jejuni in feces, and to study the distribution of C.jejuni in GI tract in a time series after gavage.Method: Recover and culture the GBS related C.jejuni strain storaged in-80℃ refrigerator in 42℃ microaerophilic environment(5%O2,85%N2,10%CO2) as the inoculation bacterium.3 to 4-weeks-old male KM mice bought from the Experiment Animal Center of Hebei Medical University were divided into four groups randomly.Group A:to investigate the existent interval of unbioactive micromolecule in KM mice feces,divide 4-weeks-old KM male mice into 3 subgroups. group A1 to A3 included 7,8 and 2 mice separately.The mice inA1 and A2 group were gavaged by 0.5 ml 140% Ba SO4 suspension,andthe mice in A3 group(control group) were gavaged by 0.5ml Brucella broth.The feces of each mice were collected in a series of time after inoculation.The transparency of feces were observed by X-ray image system to reveal the existent interval of Ba SO4 in feces.Group B:to investigate the influence of inoculating concentration ofC.jejuni on the existent interval of C.jejuni in feces of KM mice,dividethe mice into 4 subgroups,each experiment subgroups(B1-B3) had 5 mice separately and the control group(B4) had 3 mice.The inoculation concentration of each experiment group was 104CFU/ml, 108CFU/ml and 108CFU/ml separately,and the mice in control group were given clean Brucella broth.Each mice were gavaged only 1 time by 0.5ml.The feces were collected 5,10,24,30,48,72,96 and 168 hours after gavage.Separating C.jejuni in the feces to determine the existent interval of C.jejuni. The micewere sacrificed 7 days after gavage to observe the gross lesion of GI tract induced by C.jejuni.Group C:to investigate the influence of inoculating frequency,age ofmice and usage of PT on the existent interval of C.jejuni in feces of KM mice,divide the mice into 6 subgroups,each experiment subgroups(C1-C5) had 5 mice separately and the control group(C6) had 3 mice.The mice were 4-weeks-old, gavaged(3×108CFU/ml,0.5ml) 2 times in subgroup C1;4-weeks-old,given PT(200ng intraperitonially,2 times per a mice)and gavaged 1 time in subgroup C2;3-weeks-old gavaged1 time in subgroup C3; 3-weeks-old,gavaged 2 times in subgroup C4;3-weeks-old,given PT(200ng intraperitonially,2 times per a mice)and gavaged 1 time in subgroup C5.The control group were 4-weeks-old and gavaged with BrucellaBroth.Feces were collected and cultured for C.jejuni 48,72,96 and 168 hours after last gavage to determine the existent interval of C.jejuni.Mice were sacrificed 7 days after last gavage to observe the gross lesion of GI tract.Group D:to investigate the distribution of C.jejuni in KM mice’s GI tract,divide the mice into 3 subgroups, subgroup D1 and D2 had 4 mice separately,subgroup D3(control group) had 2 mice.The mice in subgroup D1 and D2 were gavaged with C.jejuni(0.5 ml 3×108CFU/ml) for 1time,and control mice were gavaged with Brucella broth(0.5ml).Mice insubgroup D1 and D2 were sacrificed 48 and 120 hours after gavage separately.GI tissues were aseptically collected, sectioned and cultured for C.jejuni.The mice in D3 subgroup were sacrificed 48 and 120 hours randomly after gavage,and the GI tissues were cultured.During the experiment,the synptoms and signs of mice were recorded,the feature of feces were evaluated by Bristol Stool Scale,and the morphology of C.jejuni succesfully separated were evaluated by Gram stain.Results:GBS related C.jejuni was succesfully recovered from-80℃refrigerator,which had positive Gram stain and hippurate hydrolation test results.The existent interval was 2-23 hours for Ba SO4,5-24 hours for C.jejuni(104CFU/ml for 1 time),5-30 hours for C.jejuni(108-1010CFU/ml for1 time) in feces of KM mice. C.jejuni were separated in the feces collected 48 hours after last gavage in subgroup C1,C3 and C5, 168 hours after last gavage in subgroup C3,and were separated in intestinal and cecal tissues 48 hours after gavage.The gross observation of inoculated KM mice’s GI tracts didn’t reveal any obvious lesions such as ulcer,edema and hemorrhage.Part of separated C.jejuni became shorter and more curved.The morphology of feces were snake,sausage and blob-shaped,no watery or mushy stool were observed.death,diahhrea or paralysis was not observed during the experiment.Conclusions:1 KM mice from the Experiment Animal Center of Hebei Medical University are not regularly gastrointestinal colonised by C.jejuni.2 The existence interval of C.jejuni in feces of KM mice after gavaged with GBS related C.jejuni for 1time is 5-30 hours,inoculating concentration, inoculating frequency and age of mice can affect the interval.3 C.jejuni can invade intestine and cecum of KM mice.4 The morphology of C.jejuni changes after passage througe the GItract. PART II The animal model of peripheral nerve damage induced by gastrointestinal infection of campylobacter jejuni and intra-peritoneal injection of pertussis toxinObject:To observe the dynamic change of mobility and body weight for KM male mice gavaged with GBS related C.jejuni and injected with PT intraperitoneally within 56 days after inoculation,and to build an animal model for peripheral nerve injury caused by naturally C.jejuni infection preliminarily.Method: Recover and culture the GBS related C.jejuni strain storaged in-80℃ refrigerator in 42℃ microaerophilic environment(5%O2,85%N2,10%CO2) as the inoculation bacterium.4-weeks-old KM male mice bought from the Experiment Animal Center of Hebei Medical University were divided into four groups randomly.Group E1 contained 20 mice,gavaged with C.jejuni for 12 times(3×108CFU/ml,0.5ml, on day1-3,8-10,15-17 and 22-24),and injected with PT intra peritoneally for 2 times(200ng,on day1 and day7);Group E2 contained 20 mice,gavaged with C.jejuni(same as E1), and injected with normal saline for 2 times(0.5ml,on day 1 and day 7);Group E3 contained 12 mice,gavaged with Brucella broth for 12 times(0.5ml,on day1-3,8-10,15-17 and 22-24),and injected with PT(same asE1);Group E4 contained 12 mice,gavaged with Brucella broth 12 times(same as E3),and injected with normal saline(same as E2).6 mice in each group were selected randomly for the observation of body weight and motility.Record the body weight from 2 days before the first gavage to day 5 every 1±1 days to reveal the short-term trendsfor weight change.Record the body weight from day 5 to day56 every7±2 days to reveal the long-term trends for weight change. Record therun time on fatigue apparatus from day 5 to day56 every 7±2 days toreveal the trends for motility change.Using Weavers scale to evaluate the sympoms and signs of mice, and using Bristol stool scale to evaluate the feature of feces.Animals were sacrificed on day14,28,42 and 56(5,5,3 and 3 mice for Group E1-E4), which sacrificed on day14 only gavaged for 6 times.Bilateral sciatic nerves and brachial plexus were extracted and fixed in 4% paraformaldehyde solution or 4% glutaraldehyde solution in 4℃ refridge for further pathological study.Results: GBS related C.jejuni was succesfully recovered from the-80 ℃ fridge.All the mice scored zero by Weavers score during the experiment,watery or mushy stools were not observed.The body weight of KM mice increased unlinear during the day before first gavage to day5.Using ANOVA analysis and LSD test to compare the body weight in each time point,the mean body weight of GroupE1 was lower than the other group(P<0.01).The body weight of KM mice increased unlinear in the first 4 weeks,waved in the second 4 weeks during the experiment.The mean body weight of group E1 was lower than group E4 except on day 13,lowerthan group E3 except on day13 and day47.The mean body weight of E2 group was lower than group E4 except on day 22,47 and 54. The body weight of E3 group equalled group E4 except on day 38 and 47.Using Kruskal-Wallis test and Nemenyi test to compare the run time difference on each time point, run time of E1 group is lower than other groups from day13 to day25(P<0.05).There were no significant different in run time among the other group on each time point.320 nerve samples were preserved in 4℃ fridge for further study.Conclusions:Gavage with GBS-related C.jejuni combining intraperitoneal injection with PT causes the decrease of mobility and the retard ofbody weight gain in an early phage. PARTIII The classification of ischemic stroke in the very elderly peopleObject:To investigate the proportion of each stroke subtype in the elderly aged 80 and over retrospectively,and analyse the discrimination of neurological function impairment on admission and the short-term prognosis at discharge among different stroke subtypes.Method:The clinical data of patients diagnosed as acute ischemic stroke,aged 80 and over and consecutively hospitallized in the neurologyward of the Eastern District of Second Hospital of Hebei Medical University were collected from Sep 14 th, 2013 to Sep 14 th, 2014. Using Oxfordshire Community Stroke Project Subtype Classification(OCSP subtype) and Trial of ORG 10172 in Acute Stroke Treatment Subtype Classification(TOAST subtype),at least 2 trained researchers separately classified the patient into a specific subtype.If there was a controversy of the classification,the final judgement was made by a chief physician of neurology. National Institute of Health stroke scale(NIHSS) score on admission and at discharge,( Barthel Index)BI score at discharge and modifiedRankin scale(m RS)score at discharge were compared by Kruskal-Wallis test and Nemenyi test among different stroke subtypes.Result:1 There were 3446 patients hospitalized during the investigation interval,in which 133 aged 80 and over,69 suffered an acute ischemic stroke( mean age 83.6±3.6,male ratio 1.02:1).2 The proportion of each subtype were 24.64%(TACI),28.99%(PACI), 21.74%(POCI) and 24.64%(LACI) for OCSP subtype;18.84%(LAA),4.35%(CE),SAO(8.70%) and 68.12%(SUE) for TOAST subtype.3 NIHSS score on admission(P<0.05) and at discharge(P<0.05) were significantly different among OCSP and TOAST subtypes, and the score variation hospitalized(P<0.05) were significantly different among OCSP subtype.The m RS score and BI score were significantly different among OCSP and TOAST subtypes(P≤0.01).Conclusion:The proportion of SUE subtype is high in acute ischemic stroke patients aged 80 and over.Neurological function impairment hospitalized and short-term prognosis of these patients vary among different OCSP and TOAST subtypes.