| BackgroundNow that intrauterine infection secondary to maternal infection is the main reason of fetal malformations suffer nervous system, and abortion, as well as neurological dysfunct-ion after birth, caused by viral and bacterial infections, have been reported a lot, so chronic brain inflammation may be one of the important pathological features associated, meanwh-ile the microglia research on nervous system inflammation in recent years has become hot. Microglia is extremely sensitive to changes which can affect the steady state of the central nervous system, likely to be activated in many neuropathological states. The microglia which be activated can release many cytokines which have function in brain injury, such as TNF-α,IL-1β,et al.The microglia’s activation may have some correlation with epithelial growthfactor receptor (EGFR) pathway which may promote its activation, thus activated microglia release associated inflammatory factor which causes damage to the central nervous system. LPS is the most common and classic inflammatory agent, is one of the microglia-activating factor identified, and repeatedly used to make inflammatory injury model of nerve cells. C225, the EGFR-specific monoclonal antibody, can inhibit of EGFR pathway activation by immune activation, so we used it as EGFR inhibitors in the intervention group of this study. This study explored the relations of activation of microglial cells inflammation-related and the EGFR pathway activation by establishing animal model, the effects of blocking the EGFR pathway on the activation of microglia inflammation-related, and the impact of EGFR inhibitors on the central nervous system injury about inflammation caused by intrauterine infection and damage repair.ObjectiveThis study was to establish an LPS animal model of pregnant rats. The pregnant rats were divided into several groups by randomization, after C225 or equal amount saline were given to them respectively, we injected LPS by intraperitoneal to simulate pregnant rats with intrauterine inflammation, further, we detected microglial activation and the changes of downstream inflammatory factors level caused by EGFR pathway regulation, finally, we speculated how the EGFR inhibitors affect the brain inflammation of intrauterine infection pups. This study aimed at exploring the relations of activation of microglia inflammation-related and the EGFR pathway activation by establishing animal model, the effects of EGFR inhibitors on the activation of microglia inflammation-related, and the impact of EGFR inhibitors on the central nervous system injury about inflammation caused by intrauterine infection and damage repair.MethodsWe put 80 female rats and 40 male rats into a cage at 20:00 one day, and checked the vaginal smears to look up sperm to see whether they were pregnant or not, the next day at 8 am. The female rats found sperm in vaginal smears were considered to be pregnant, and put them into another cage. The pregnant female rats were divided into intervention group (C225+LPS group)、model group(LPS group) and control group by randomization. LPS lyophilized preparation was dissolved in sterile saline to formulate to 1g/ml and 0.5g/ml in two sizes. The solution was shaken to be mixed, and then 4 ℃ saved backup. When pregnant female rats’pregnancy was 14 days, the model group of pregnant rats had a injection at a concentration of 0.5g/ml LPS solution 0.2ml, and the intervention group pregnant rats had a injection at a concentration of 1.0g/ml LPS solution 0.1ml and addition of 0.1ml sterile saline containing 250 nM C225, the control group of pregnant rats had a injection of sterile saline 0.2ml, continuous injection of 5 days. We selected six newborn rats from each group and raised them to twenty-eight days old, then we would understand their learning and memory ability through the morris water-maze test.1,3,5 days after the birth of newborn rats were taken to weigh, and part to be done brain paraffin, the other part were decapitated directly, whose brain specimens from fresh brain tissue samples were stored in liquid nitrogen and the next day were transferred to and stored at-80℃ refrigerator.TTC staining to observe the the gross morphology of brain tissue. We did them with HE staining and observed their pathological changes, and detected the expression of OX42 and pEGFR (Phosphorylation of growth factor receptor) by immunohistochemical methods. Expression levels of Microglia activation products TNF-α and IL-1β were detected by ELISA. We took the plancents of the pregnant rats from each group to do HE staining,and observed their pathological changes. The results were statistically analyzed using SPSS 19.0, and P<0.05 was considered statistically significant.Result1.Compared with the model group at the same time, the weight of newborn rats compared withthe intervention group and the control group rats were lower, and the difference was statistically significant (P<0.05);The newborn rats of intervention group were lighter than the control group rats, and the difference was statistically significant (P <0.05); The weight of the newborn rats from intervention group and the control group growthed fast than the model group,but there was no difference between the intervention group and the control group.The water maze test showed that the average escape latency of the model group was longer than the intervention group and the control group, and the differences were statistically significant (P<0.05); the intervention group was longer than the control group, and the difference was statistically significant (P<0.05)2.TTC staining showed that there were lesions in the brain tissue of newborn rats from the model group. HE staining of brain tissue showed that the model group of pregnant rats cells swelled, disordered, and accompanied by the disappearance of neurons; Intervention group of normal brain cells are still visible structure, cellular level is still distinct, partially visible infiltration of inflammatory cells; Brain tissue structure of the control group clearly structured as well as cells.HE staining of the placents showed that there was neutrophil infiltration in the model group and the intervention group,but the the structure of the placents from the control group was clear, without inflammatory cellular infiltration.3.1mmunohistochemistry results showed that there were more positive-cells expressing OX42 and pEGFR in the brain tissue of the model group and the intervention group of pregnant rats than the control group, and the difference was statistically significant (P<0.05); there were more positive-cells expressing OX42 and pEGFR in the brain tissue of the model group of pregnant rats than the intervention group, and the difference was statistically significant (P<0.05).In group comparison,3 and 5 days group had more positive-cells expressing OX42 and pEGFR than 1day group, and the difference was statistically significant (P<0.05); Between 3days and 5days group, there were almost same OX42 and pEGFR expressed, and the difference was not statistically significant (P> 0.05). While we compared the intervention group and the control group in each group comparision,OX42 and pEGFR had no difference in the amount of positive expression at each time point, and the difference was not statistically significant (P>0.05)4.ELISA assay showed that there were both more TNF-aand IL-1β in the brain tissue of the model group of pregnant rats than the intervention group (P<0.05), and the difference was statistically significant; There were more TNF-ain the brain tissue of the intervention group of pregnant rats than the control group, and the difference was statistically significant (P<0.05); While there were almost same IL-1βexpressed between intervention and control group, and the difference was not statistically significant (P> 0.05).In model group,3days and 5days group had more TNF-αand IL-1βthan 1days group, and the difference was statistically significant (P<0.05),but there were no difference between 3days and 5days group;At each time points,therewere no difference in the intervention group and the control group on the cotent of TNF-aand IL-1β (P>0.05)Conclusion1. It confirmed that intrauterine infection in pregnant rats modeling were established successfully through weight monitoring and pathological observation over damaged brain tissue of rats which were given birth by model group of pregnant rats.2. The expression of activated microglia and the content of TNF-a and IL-1β of the brain tissue from the intervention group were lower than the model group.We infered that there may be a relationship between EGFR pathway and the activation of microglia.3. Using EGFR inhibitors C225 intervene the intrauterine infection in pregnant rats modeling could could reduce the damage of newborn rats brain tissue, playing a neuroprotective role.4. EGFR inhibitors C225 may reduce the incidence of brain injury caused by being exposed in utero inflammation. |
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