| Objective:White mater damage (WMD) in the premature babies is considered as the most common and important form of injury to the preterm brain. Preoligodendrocyte (preOLs) is the key target cell of WMD. Recently, arrest of preOLs differentiation was reported to be involved in WMD. The epidemiologic evidence and study on animal models supported the view that one of the most important factors that associated with WMD appeared to be intrauterine infection. Neural immune response has long been considered to play an important role in the WMD induced by intrauterine infection. But the exact molecular mechanism is still unclear.The identification and the study of Toll-like protein is one of the most advances in the field of inflammatory signal transduction. Activation of TLRs recruits downstream signaling proteins, results in transcription of genes encoding inflammation-associated molecules. TLRs control innate immune response and finally initiate adaptive immune response. A series of studies pointed out that TLR-4 signal pathway was involved in many brain injury associated with immune response such as ischemic stroke, Parkinson disease, multiple sclerosis, and so on. Thus, some researchers proposed a key role of the activation of TLR-4 signal pathway in the balancing mechanism of the hurt and the defense in brain upon stimulation. What is more, some studies confirmed lately that activation of TLR-4 signal pathway can directly induce expression of canonical notch target genes. Notch1 signal pathway disturbs the differentiation of oligodendrocytes by inducing the expression of the downstream target genes such as Hairy enhancer of split gene 5 (Hes5).Now, few researches focused the effect of TLR-4 signal on the WMD induced by intrauterine infection especially in vivo level. In past several years, our laboratory has successfully established an animal model of neonatal rat WMD induced by intrauterine infection after endocervical inoculation with Escherichia coli (E. coli) suspension. On the basis of this stable animal model, we observed the expression of TLR-4 signal molecules in the developing brain (including fetal rats and neonatal rats) after intrauterine infection in our present study. To make further efforts to investigate the potential mechanism by which TLR-4 signal contributes to the development of the WMD after intrauterine infection, we detected the expression of TLR-4 associated downstream cytokines and negative regulatory factors during preOLs maturation. This study may offer lots of experimental information and evidence on the clinical diagnosis and treatment of preterm WMD, may constitute a solid experimental basis in "in vivo" level for further study in correlative field, and may provide new targets for the future treatment and prevention of preterm WMD.Methods:1. Group:All rats were randomly divided into control group and intrauterine infection group.In the intrauterine infection group, pregnant rats at 15 days of gestation were inoculated endocervically with 0.4 mL of E.coli suspension. While in the control group, the rats were injected endocervically with 0.4 mL of sterile saline solution instead.2. Sample collection:The developing rats(including fetuses delivered by cesarean section and neonates) were sacrificed by decapitation at embryonic day 17(E17), E19, E21 and postnatal day 1 (P1),P3 and P7, P14, and the brains were fixed in neutral formaldehyde or stored at-80℃freezer.3. Experimental methods:The body weight and brain weight of all the developing rat in both groups were recorded. Hematoxylin-eosin (HE) staining determined the cerebral WMD of neonatal rats at P1, P3, P7 and P14. Western blot analysis were used for evaluation of myelin basic protein(MBP), TLR-4 and nuclear factor-kappa B p65 (NF-κB p65) protein levels in developing rat brains at E17, E19, E21, P1, P3, P7, P14. Real-time quantitative RT-PCR was used to analyze TLR-4, NF-κB p65, tumor necrosis factor (TNF)-a, interleukin (IL)-1β, as well as Notch 1, Hes5 mRNA expression in developing rat brains at E17, E19, E21, P1, P3 and P7.Results1. Forty-four dams had normal food intake and activity, and none died or delivered immature fetuses after inoculation. In the control group,66 neonatal rats,56 fetal rats of E17,55 fetal rats of E19,58 fetal rats of E21 were collected. There were 7 stillborn fetuses,2 stillborn neonates in the control group. In the intrauterine infection group,70 neonatal rats,57 fetal rats of E17,58 fetal rats of E19,56 fetal rats of E21 were collected. There were 7 stillborn fetuses,2 stillborn neonates in the intrauterine infection group. Inflammation of the uterus and the placenta was induced after intrauterine E. coli inoculation, but all cases of the control group had no histologic evidence of intrauterine infection.2. After intrauterine infection, the body weight and brain weight of E17, E19, E21 fetal rats and P1 neonatal rats significantly decreased.3. HE staining procedures revealed clear staining and normal structure of periventricular white matter from P7, P14 rats of the control group. Weak staining and focal rarefaction of periventricular white matter were shown in P7, P14 rats of the the intrauterine infection group.4. The expression of cerebral MBP was detected in P14 rats of the control group, while the expression of MBP in the brain of P14 rats decreased or disappeared after intrauterine infection.5. The expressions of cerebral TLR-4 and downstream signal molecule NF-κB p65 increased significantly in phase after intrauterine infection at E17, P1, P3, P7.6. The expression of cerebral TNF-a mRNA was up-regulated at P19, P21 after intrauterine infection.7. The expressions of cerebral Notchl and downstream signal molecule Hes5 increased significantly in phase after intrauterine infection at E19, P1, P3, P7.Conclusion:1. Intrauterine infection decreased the body weight, the brain weight of fetal rats.2. TLR-4 signal might play a certain role in the development of the WMD induced by intrauterine infection.3. TLR-4 signal might contribute to the development of the WMD after intrauterine infection partly by regulating the expression of certain cytokines such as TNF-a.4. TLR-4 signal might contribute to the development of the WMD after intrauterine infection partly by activating Notchl signal that arrested the differentiation of preoligodendrocytes.
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