| Objective: To measure the IL-6, IL-8levels in animal model of amniotic fluid embolism,and explore the pathogenesis of amniotic fluid embolism.Methods:1.Established the rat model of amniotic fluid embolismThe pregnant rats were randomly divided into three groups, the Control group, theExperiment group1(Amniotic liquid group) and the Experiment group2(Meconiumamniotic fluid group), n=10. After the anesthetic works, the hysterectomy surgery wereperformed, then the peritoneal were closed. The left common carotid artery were separatedand Intubated, the tee were Turned and connected with the Two-channel physiologicalrecorder, to monitor the hemodynamic indexes continuously. The prepared injection wereinjected into the abdominal veins, dose of2.5ml/kg. The blood (1ml) extracted from theleft common carotid artery before injection60min and after injection60min. The bloodwere extracted if the rats had died immediately in the observation process. The blood wascentrifuged10min(3000r/min), the supernatant was stored in the-20°C refrigerator. Theleft lung tissue were used for pathological examination.2. The preparation of supernatant of meconium amniotic fluidThe abdominal cavity of rat were opened, and the intestine were removed, themeconium were squeezed gently and grounded with a homogenizer, then were formulatedinto1%~7%meconium amniotic fluid, centrifuged10min(3000r/min).All data was processed using SPSS18.0, T test was used within groups, the pairwisecomparison with multiple sample mean was used between groups. P <0.05was statisticallysignificant.3. The IL-6detection methodThe IL-6detection method is using the enzyme-linked immunosorbent assay (ELISA).A known concentration of IL-6standard, unknown concentration of sample was added tothe micro ELISA plate for testing. Firstly, IL-6and antibody labeled with biotin were incubated simultaneously. After washing, streptavidin labeled HRP was added. Afterfurther incubation and washing, to remove unbound enzyme conjugate and then added thesubstrate A, B, and effected with conjugate simultaneously, then color appeared. The colordepth showed a proportional relationshipa with the sample concentrations of IL-6.4. The IL-8detection methodThe IL-8detection method is using the enzyme-linked immunosorbent assay (ELISA).A known concentration of IL-8standard, unknown concentration of sample was added tothe micro ELISA plate for testing. Firstly, IL-8and antibody labeled with biotin wereincubated simultaneously. After washing, streptavidin labeled HRP was added. Afterfurther incubation and washing, to remove unbound enzyme conjugate and then added thesubstrate A, B, and effected with conjugate simultaneously, then color appeared. The colordepth showed a proportional relationshipa with the sample concentrations of IL-8.Results:1.The rat model of amniotic fluid embolism was established successfully, the clinicalmanifestations were more serious in the experiment group1and experiment group2, therewas no deaths of pregnant mice within60min in the experiment group1and one deathswithin60min in the experiment group2. The results of peripheral hemodynamic:Hemodynamic indicators in the control group had transient change and was not statisticallysignificant（P>0.05）, the arterial systolic, diastolic, and mean arterial pressure decreasedsignificantly in experimental group1and the experimental group2compared with thecontrol group（P<0.05）, which was most obvious10min after injection of amniotic fluidand maintaining a low level of state30min to60min, the mean arterial pressure declinedpersistently to approximately20mmHg in a pregnant rat in experiment group2and heartfailure happened finally. There was no significant difference and between experimentalgroup1and experimental group2, there was no significant difference（P>0.05）of the heartrate changes in three groups of pregnant rats.2.The concentration of IL-6:(20.38±7.73)μg/ml before injection and (19.47±6.46)μg/mlafter injection in Control group,(21.41±6.47)μg/ml before injection and(30.74±5.61)μg/ml after injection in Experiment group1,(20.27±5.51)μg/ml beforeinjection and (36.44±5.47)μg/ml after injection in Experiment group2.3.The concentration of IL-8:(15.78±4.36)μg/ml before injection and (15.29±4.76)μg/ml after injection in Control group,(16.21±3.89)μg/ml before injection and(23.02±8.06)μg/ml after injection in Experiment group1,(15.49±4.25)μg/ml beforeinjection and (38.8±9.02)μg/ml after injection in Experiment group2.4. The results of HE staining showed that there was varying degrees of pulmonaryedema, inflammatory cell infiltration, squamous epithelium, mucus and other amnioticfluid components in the pulmonary vascular in experimental group1and experimentalgroup2, there was no no significant change in the control group.5.The results of CK10Immunohistochemistry staining showed that there was squamousepithelium and other amniotic fluid components in the pulmonary vascular in experimentalgroup1and experimental group2, there was no no significant change in the control group.6. The results of APM staining showed that keratinized squamous epithelium was pink,mucus in the lumen of small bronchial was blue, neutrophil cytoplasmic was light blue orpink, there was no no significant change in the control group.Conclusion:1. The severity of the clinical manifestations of pregnant rats was related thecomponents into the mother’s amniotic fluid, AFE should be alerted when meconiumcontamination occurs.2. The IL-6content increased in Experiment group, and significantly in Meconiumamniotic fluid group, which showed that some substances of meconium could stimulate thebody to produce large amounts of IL-6.3. The IL-8content increased in Experiment group, and significantly in Meconiumamniotic fluid group, which showed that some substances of meconium could stimulate thebody to produce large amounts of IL-8. |
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