| White mater damage (WMD) in the premature babies is considered as the most common and important form of injury to the preterm brain. Characteristically, damage is localized to white matter, involving both a diffuse astrogliosis with subsequent loss of myelinproducing oligodendrocytes as well as multifocal necrosis resulting in cystic change (periventricular leukomalacia, PVL). Clinical and epidemiological studies have shown significant associations among intrauterine infection/inflammation, premature and preterm WMD. The activation of both glial cells and inflammation signal play an important role between intrauterine infection/inflammation and perinatal brain damage. Premature astrocyte proliferation and astrogliasis affected by inflammatory reaction after intrauterine infection/inflammation was the major reason for WMD. Toll-like receptor-4/Nuclear factor-KB (TLR-4/NF-κB) and Notchl/Hes might play a key role on premmature astrocyte proliferation and astrogliosis after intrauterine infection/ inflammation. Thus, in order to investigate the spatio-temporal characteristics of premature astrocyte proliferation and astrogliosis in the developing rat brain after intrauterine infection/inflammation, we developed a model of intrauterine infection in rat to observe the identification of glail fibrillary acidic protein (GFAP). Moerover, astrocytes were isolated from cultures of fetal rat brains, and the astrocyte proliferation and astrogliosis after infection/inflammation was analyzed, and the role of TLR-4/NF-κB and Notchl/Hes signalling pathway were detected. Furthermore, to determine the effect of tea polyphenol-Epigallocatechin gallate (TP-EGCG) to premature astrocyte proliferation and astrogliosis, the astrocytes were treated with TP-EGCG to detect the characteristics of astrogliosis, and we study whether the inhibition of TLR-4/NF-κB and Notchl/Hes signalling pathway by TP could influence the astrocyte proliferation and astrogliosis. Taken together, the present research will provide new targets for the future treatment and prevention of preterm WMD.Objective:To study the spatio-temporal characteristics of astrocyte proliferation and astrogliosis in different brain region (periventricular white matter, hippocampus, corpus callosum and cerebral cortex) at different time after intrauterine infection/inflammation, and to investigate the characteristics of astrocyte proliferation and the expression of TLR-4/NF-κBp65, cytokine(TNF-a, IL-1p, MIP-1α, MIP-1β, etc.), Notchl/Hes after infection/inflammation, and to study the effect of TP-EGCG to premature astrocyte proliferation and astrogliosis and the role of TLR-4/NF-κB and Notchl/Hes signalling pathway.Methods:1. Animal model: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 0.2 mL of E.coli suspension. While in the control group, the rats were injected endocervically with 0.2 mL of sterile saline solution instead. Pups from each of the two groups were decapitated on postnatal day 1 (P1), P3, P7, P14 and P21 and the brains were immediately collected, and the body weight and brain weight of the pups in both groups were recorded. Histological characteristics of the cerebral WMD of pups were studied by hematoxylin-eosin (HE) staining and immunohistochemistry was used for evaluation of GFAP expression in pup brains at P1, P3, P7, P14 and P21.2. Cell cultures:Immunofluorescence methods were used for evaluation of GFAP+/BrdU+expression after incubated for different times(l,3,7days) with different concentrations(lμ.g/mL,5μg/mL, lOμg/mL) of lipopolysaccharide(LPS), and the concentrations of cytokines(TNF-a, IL-1β, IL-10, etc.) in the samples of culture medium were analyzed by Enzyme-linked immunosorbent assays (ELISA). Moreover, real-time quantitative RT-PCR was used to analyze TLR-4, NF-κB p65, tumor necrosis factor (TNF)-a, interleukin (IL)-1β, Macrophage inflammation protein(MIP)-lα, MIP-1β, as well as Notchl, Hesl, Hes5 mRNA expression in astrocytes after exposed to LPS.3. TP-EGCG treatment:Immunofluorescence methods were used for evaluation of GFAP+/BrdU+expression after incubated for different times(1,3 days) with LPS(5μ,g/mL) and TP-EGCG(1mM), real-time quantitative RT-PCR was used to analyze TLR-4, NF-κB p65, TNF-a, IL-1β, MIP-lαMIP-1β, as well as Notchl, Hesl, Hes5 mRNA expression in astrocytes after exposed to LPS and TP-EGCG.Results:1. 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. After intrauterine infection, the body weight and brain weight of P1 neonatal rats significantly decreased (P＜0.05). At P1 and P3, GFAP was expressed very scarcely in periventricular white matter but not in other brain regions between the two groups (P>0 05). Compared with the control group, at P7 GFAP expression of the E.coli-treated pups was remarkably increased in periventricular white matter and hippocampus (P＜0.05). The E.coli-treated pups at P14 showed a marked increase of GFAP expression in peri ventricular white matter, corpus callosum and cortex (P＜0.01). However, no significant difference in levels of GFAP expression in any brain regions were found at P21 between the two groups (P>0 05).2. Compared with the control group, the numbers of GFAP+/BrdU+cells were significantly decreased after exposed for 1 and 3 days with LPS (l0μg/mL) (P＜0.05), and the numbers of GFAP+/BrdU+cells were significantly increased after exposed for 3 and 7days with LPS (5μg/mL) (P＜0.05). The concentrations of cytokines (TNF-a, IL-1β) of culture medium were remarkably increased after incubated early with LPS (5μg/mL) (P＜0.05). TLR-4 mRNA expression of the LPS (lμg/mL)-treated astrocytes was lower than the control after incubated for 1 day (P<0.05). However, TLR-4 mRNA expression of the LPS (5μg/mL)-treated astrocytes was higher than the control after incubated for 3 day (Pμ＜0.05). The expression of NF-KBp65 mRNA was decreased after exposed for 3 days with LPS (1μg/mL) (P＜0.05), and after exposed for7 days with LPS (5μg/mL and lOμg/mL), NF-κBp65 mRNA expression was increased (P＜0.05). The expression of Notchl mRNA was decreased after exposed for 3 and 7 days with LPS (lμg/mL) (P＜0.05), and after exposed for lday with LPS (lOμg/mL) and 7 days with LPS (5μg/mL), Notchl mRNA expression was increased (P＜0.05). Moreover, The expression of Hesl mRNA was decreased after exposed for 1 day with LPS (lOμg/mL) and 7 days with LPS (1μg/mL) (P＜0.05), however, after exposed for 1 and 3 days with LPS (1μg/mL), Hes5 mRNA expression was increased (P＜0.05).3. Compared with the LPS-treated group, the numbers of GFAP+/BrdU+cells were significantly decreased after treated with TP-EGCG (P＜0.05), and no significant difference was found between the LPS+EGCG group and the control group (P>0.05). The expression of TLR-4 mRNA showed no difference between the LPS+EGCG group and the LPS group (P>0.05), but NF-κBp65 mRNA expression was remarkably decreased in LPS+EGCG treated astrocytes (P＜0.05). There were no differences in the expression of TNF-αIL-1β、MIP-1α、MIP-1βmRNA between the LPS+EGCG group and the LPS group (P>0.05). Moreover, There were significant differences in the expression of Notch1、Hes1 mRNA between the LPS+EGCG group and the LPS group (P<0.05), but the expression of Hes5 mRNA showed no difference between the two groups (P>0.05).Conclusion:1. The spatio-temporal characteristics of GFAP expression in the developing rat brain after intrauterine infection/inflammation indicated that intrauterine infection/ inflammation might alter the astrocytic function and damage the developing white matter.2. The characteristics of premature astrocyte proliferation and astrogliosis were different under different infection/inflammation condition. The transient increase in expression of cytokine such as TNF-α, IL-1β, MIP-1α, MIP-1β, IL-10 were found in astrocytes after LPS treated, and the expression of TLR-4/NF-κB and Notchl/Hes in astrocytes were also different under different infection/inflammation condition.3. TP-EGCG could alter the characteristics of premature astrocyte proliferation after LPS treated, but they could not alter the expression of TLR-4 mRNA and cytokine in LPS-treated astrocytes. Moreover, TP-EGCG could alter NF-κBp65 mRNA expression in astrocytes under infection/inflammation condition, and they also had an effection on the expression of Notch1, Hes1 mRNA in astrocytes under infection/ inflammation condition. |
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