| PART I Graded doses(0.1-20?g) of pertussis toxin is dose-independent of the peripheral neuropathy rate of Hartley Guinea pig induced by CjObjective:Although Pertussis toxin can promote the formation of peripheral neuropathy model of Hartley Guinea pig caused by Cj, the rang of dose is still unknown. Investigate the influence of grades doses(0.1-20?g) of pertussis toxin on the peripheral neuropathy rate of Hartley Guinea pig induced by Cj.Methods:1 Cj suspension preparation: We selected Lulei strain from the library of C.jejuni strains as experimental strains,which were incubated under laboratory standard micro-aerobic conditions(42?,10%CO2?5%O2?85%N2) in blood agar.They were purified and suspended to concentration of 3 × 108 CFU/ml.2 Pertussis toxin injection preparation:Adding 50?l distilled water to 50?g pertussis toxin powder in order to make preparation for different concentrations of pertussis toxin injection.3 Grouping method:12 SPF Hartley guinea pigs were divided into 5 experimental groups and 1 control group randomly,2 per group.Group A: 0.1?g pertussis toxin+3 × 108 CFU/ml Cj; Group B: 1?g pertussis toxin+3 × 108 CFU/ml Cj; Group C: 2?g pertussis toxin+3 × 108 CFU/ml Cj; Group D: 10?g pertussis toxin+3 × 108 CFU/ml Cj; Group E: 20?g pertussis toxin+3 × 108 CFU/ml Cj; Group F: no treatment.4 Operating method: Guinea pigs were fasted for 15 hours, deprived of water for 3 hours before injected with corresponding dose of pertussis toxin. Fed them with bacterial suspension the next day.5 Clinical symptom observation and anatomy: Guinea pigs were observed everyday and weighed regularly. Clinical symptom refer to the evaluation criteria of Hartley guinea pig in EAE model: There are 5 grades according to paralysis degree. Grade 0: normal; Grade 1:toes of hind legs drag slightly; Grade 2:toes of hind legs drag obviously without involving other parts; Grade 3: hind legs drag itself so obvious that incline to one side; Grade 4: hind legs paralyse completely; Grade5: quadriplegia, even respiratory depression.Experimental animals were dissected 4 weeks after the poison attack.One of each group was dissected after anesthesia without perfusion.Their left sciatic nerves were soaked in 4%paraformaldehyde and then be dyed by osmium tetroxide.The right ones were dyed by HE and Gomori after been frozened. The other one of each group was dissected after been perfused by 4%paraformaldehyde. Both sides of sciatic nerves were conserved in 4%paraformaldehyde and used for osmium tetroxide.6 Neuropathological observation and statistic analysis: Osmium tetroxide ?HE and Gomori staining results were observed and 100 teasing fibers of each guinea pig were examinated.Computing the incidence of neuropathological rate. Making stastic analysis to peripheral nerve lesion rates among groups and between two guinea pigs in each group.Results:1 Clinical symptom: There was no abnormal phenomenon in the control group such as poor spirit and appetite, limb activity disorder, fur dirty and so on in the whole experimental process. However, mental state and appetite of Guinea pigs in experimental groups became worse on pertussis toxin injection day and returned to normal the next day.The right hind leg of NO.8 guinea pig could not stand after pertussis toxin injection.Assess the score of clinical symptom :grade 2.Its exercise capacity become better after 5 minutes and return to normal after 30 minutes. The belly fur of NO.6 guinea pig became dirty after 17 days of poison attack. There was no limb activity disorder and fur dirty in rest of guinea pigs in experimental group; 2 Neuropathological observation: 2.1 osmium tetroxide staining result: Sciatic nerves of experimental groups appeared phenomenon as follows: demyelination, myelin fracture gathered into pieces, axon with varying thickness, axonotmesis, node of Ranvier become lengthen. Sciatic nerves of control groups appeared phenomenon as follows: Both myelin and axon were smooth, nodes of Ranvier are nomal. 2.2 HE staining result was as follows: axonotmesis, axon with varying thickness, myelin with varying degrees of vacuolar degeneration. 2.3 Gomori staining result was as follows: axonotmesis, axon with varying thickness, myelin with varying degrees of vacuolar degeneration; 3 Osmium tetroxide statistic result of peripheral neuropathy rate: Peripheral neuropathy rate of Hartley guinea pig in each group are as follows: Group A(0.1?g) 12.5%,Group B(1?g) 22.5%,Group C(2?g) 23%,Group D(10?g) 19.5%,Group E(20?g) 22%,Group F(0?g) 2.5%.There is statistical differences between group F and group A-E. No statistical differences among experimental groups. No statistical differences between dissection without perfusion and dissection after perfusion.Conclusions:1 Cj combining with PT can lead to peripheral neuropathy in Hartley guinea pigs.2 Graded doses(0.1-20?g) of pertussis toxin is dose-independent of the peripheral neuropathy rate of Hartley Guinea pig induced by Cj. PART II Influence of Cj feeding frequency to the rate of peripheral neuropathy of the Hartley guinea pigObjective:Exploring the best feeding frequency in the animal model of Cj introducing Hartley guinea pig's peripheral neuropathy in order to improve the method of model building.Methods:1 Bacterial suspension preparation: We selected Lulei strain from the library of C.jejuni strains as experimental strains,which were incubated under laboratory standard micro-aerobic conditions(42?,10%CO2?5%O2?85%N2) in blood agar.They were purified and suspended to concentration of 3 × 108 CFU/ml.2 Experimental procedure: 32 four-week-old SPF Hartley guinea pigs were randomly divided into two experimental groups and one control group, 12 in each experimental group and 8 in each control group. Experimental group A: They were fed once every three days,7 times continuously. Experimental group B: They were fed once every two days,10 times continuously. Control group C: blank controller. Three guinea pigs in each group were dissected every 5 days after the last feeding. Feeding method: Guinea pigs were feeding with 3 × 108 CFU/ml Cj, before which they were fasted for 15 hours and deprived water for 3 hours.3 Clinical symptom observation?Neuropathological observation and statistic analysis: Guinea pigs were observed everyday and weighed regularly. Clinical symptom refer to the evaluation criteria of Hartley guinea pig in EAE model(refer to part 1). They were dissected for sciatic nerve which would be stained with osmium tetroxide according to specific time. 100 teasing fibers of each guinea pig were examinated. Computing the incidence of neuropathological rate. Making stastic analysis to peripheral neuropathy rates among groups and dissection batch.Results: 1 Clinical symptom: There was no abnormal phenomenon in the control group such as poor spirit and appetite, limb activity disorder, fur dirty and so on in the whole experimental process. Mental state and appetite of Guinea pigs in experimental groups were also good.Howver, NO.16 and NO.79 were observed depilation after 5 days of the last feeding. NO.79 became normal after 18 days of the last feeding neither nor the NO16 which stayed the course; 2 Neuropathological observation: Sciatic nerves of experimental groups appeared phenomenon as follows: myelin fracture gathered into pieces, axons were compressed, myelin shriveled into Great-Wall like structure.Sciatic nerves of control groups appeared phenomenon as follows: Both myelin and axon were smooth, nodes of Ranvier are nomal; 3 Osmium tetroxide statistic result of peripheral neuropathy rate:Peripheral neuropathy rate of Hartley guinea pig in each group are as follows: Group A 24.17%,Group B 25.83%,Group C 3.125%. Peripheral neuropathy rate of Hartley guinea pig among dissection batches in each group are as follows:Group A1 19.33%, Group A2 25.67%, Group A3 26.67%, Group A4 25.00%, Group B1 21.33%, Group B2 25.00%, Group B3 26.67%, Group B4 30.33%, Group C1 3.5%, Group C2 2.5%, Group C3 3.0%, Group C4 3.5%.There is statistical differences between control group and experimental group. No statistical differences between experimental groups. No statistical differences among dissection batches in each group.Conclusions:1 Frequency increasing can't enhance the peripheral neuropathy rate of the experimental animal.2 There is no stastic difference among dissection batches since 20 days after the completing of poison.3 Campared to contact frequency,the immune state has further impact on the occurrence of body. |
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