Role And Mechanism Of CREB Signaling Pathway In Hippocampus Neurogenesis Of Neonatal Rats After Intrauterine Inflammation

Intrauterine infection/inflammation has been identified as the most common cause of preterm delivery and neonatal complications. Fetal inflammatory response syndrome (FIRS) was implicated as a cause of fetal or neonatal injury that leads to cerebral palsy (CP). White mater damage (WMD) is identified clinically as periventricular leucomalacia (PVL) which is associated with the subsequent development of impaired neurological outcomes of variable severity including CP. Neurogenesis arising from populations of neural progenitors that persist in the sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the dentate gyrus (DG) after hypoxia-ischemia are now a universally accepted feature of the adult mammalian brain. However, it remains unknown whether neurogenesis is induced by perinatal infection in the developing brain. The cAMP response element binding protein (CREB) is a transcription factor in eukaryote and plays important roles in the regulation of neurogenesis, synapse formation, learning and memory. The phosphorylation of CREB is the important way to regulate transcription, and the transcription of many target genes is activated after the phosphorylation of CREB affected by extracellular signals, showing various physiological functions. The goal of the current study is to examine the effect of intrauterine inflammation on hippocampal neurogenesis and to examine whether the CREB signaling pathway is involved in inflammation-induced neurogenesis and the associated molecular mechanism. Thus, in order to investigate the spatio-temporal characteristics of hippocampal neurogenesis in the developing rat brain after intrauterine inflammation, we developed a model of intrauterine inflammation in rat to observe cell proliferation, neuronal survival and differentiation. Moreover, the mRNA and protein expression of p-Akt,p-CREB and BDNF were analyzed. Furthermore, to determine the role and molecular mechanism of CREB signaling pathway, we study whether the inhibition of PI3K/Akt signaling pathway by LY294002and activation of CREB signaling pathway by Rolipram could influence the neurogenesis and cognitive function. Taken together, the present research will provide new targets for the future treatment and prevention of preterm WMD. The identified genetic pathways provide insight into the inflammation-induced neurogenesis that is so central in learning and memory and will direct future clinic studies on the functional rehabilitation of cerebral palsy.Objective:To examine the effect of intrauterine inflammation on hippocampal neurogenesis, and to examine whether the CREB signaling pathway is involved in inflammation-induced neurogenesis, and to study the role and associated molecular mechanism of the CREB signaling pathway in neurogenesis and cognitive function.Methods:1. Animal model:All rats were randomly divided into control group and intrauterine inflammation group. In the intrauterine inflammation group, pregnant rats at15days of gestation were inoculated endocervically with0.2mL of E. coli suspension. While in the control group, the rats were injected endocervically with0.2mL of sterile saline solution instead. Pups from each of the two groups were decapitated on postnatal day3(P3), P7, P14, P21and P28and the brains were immediately collected for further study. Histological characteristics of the cerebral WMD of pups were studied by hematoxylin-eosin (HE) staining and immunohistochemistry was used for evaluation of neurogenesis in the DG of hippocampus. To evaluate cell proliferation, intraperitoneal injections (i.p.) of BrdU (50mg/kg) were given twice daily (at12-hr intervals) for3consecutive days. Rats were sacrificed on P3, P7, P14and P28just after receiving BrdU on P1-3, P5-7, P12-14and P26-28, respectively. To evaluate neuronal survival and differentiation, the same dose of BrdU was cumulatively delivered at P1for7consecutive days. Rats were allowed to survive for21days and were sacrificed on P28. BrdU/Nestin co-labled cells were used to identify NSPs, BrdU/NeuN co-labled cells were used to identify mature neurons differentiation and BrdU/GFAP co-labled cells were used to identify astrocyte differentiation.2. Morris water maze:The water maze test was performed in a circular swim arena (diameter of100cm, height of60cm) that was surrounded by extra-maze visual cues that remained in the same position for the duration of training and was filled to cover the platform by1cm at (22±0.5)℃. Water was made opaque with nontoxic, white tempera paint. The escape platform was a circular, nonskid surface (10cm in diameter) placed in the northwest (NW) quadrant of the maze. Acquisition training consisted of5test days with four daily trials. Male rat entered the maze facing the wall and began each trial at a different entry point in a semirandom order. Trials lasted120s or until the animal mounted the platform, with a15min intertrial interval. A probe trial was conducted on day6at which time the platform was removed; the animal swam for120s and the time spent in the target quadrant (NW) versus the adjacent and opposite quadrants was recorded. A video camera mounted above the swim arena and linked to Top Scan software recorded swim distance, swim speed, and time to platform, and was used for tracking and analysis.3. CREB signaling pathway and neurogenesis:Real-time quantitative RT-PCR and Western blot were used to analyze p-Akt,p-CREB,BDNF mRNA and protein expression in hippocampus tissues corresponding to each group. Moreover, immunohistochemisty methods were used for evaluation of p-Akt,p-CREB and BDNF expression in the DG of hippocampus.4. LY294002and Rolipram treatment:The Morris water maze test was performed to evaluate their hippocampus dependent spatial learning and memory changes after LY294002and Rolipram treatment. Immunofluorescence methods were used for evaluation neurogenesis in the DG of hippocampus after LY294002and Rolipram treatment. Real-time quantitative RT-PCR and Western Blot were used to analyze p-Akt,p-CREB-BDNF mRNA and protein expression changes in hippocampus tissues after LY294002and Rolipram treatment.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 inflammation. In place navigation, rats of two groups took decreasing time over the course of the training to locate the submerged platform indicating that rats could definitely get and remember the location of the hidden platform through training and exhibit increasingly efficient exploratory strategies during the following trials. Overall repeated ANOVA analyses showed there were significant differences from day1to day3between E. coli-infected group and control group (P<0.05). And data also showed that there were no statistical differences since day4until the end of the test (P>0.05). In intrauterine E. coli infected group, there was significant increase in numbers of BrdU-labeled cells than that of the control group (P<0.05), and there was a significant increase in BrdU/Nestin coexpression in intrauterine E. coli infected group than controls (P<0.01). Despite the large increase in the total number of BrdU-labeled cells in the E. coli infected rats, no significant differences were found in the percentage of newborn cells expressing a neuronal phenotype (BrdU+/NeuN+) and glial phenotype (BrdU+/GFAP+) in these two groups (P>0.05).2. Compared with the control group, the expression of p-Akt mRNA and protein were significantly increased on P3,7,14(P<0.05), the expression of p-CREB mRNA and protein were significantly increased on P14,28(P<0.05), and the expression of BDNF mRNA and protein were significantly increased on P3,7(P<0.05). Moreover, immunohistochemisty showed p-Akt,p-CREB and BDNF had expression in the DG of hippocampus.3. The ANOVA analyses showed that rats of LY294002group failed to locate the platform in the target quadrant. Percentages of time in the target quadrant and numbers of platform crossings were less in LY294002group than those of control group (P<0.05), however there were prominent improvements in LY294002+Rolipram group especially quadrant dwell time (P<0.05). Meanwhile, there was no significant difference in the target quadrant and numbers of platform crossings between LY294002+Rolipram group and control group (P>0.05). Compared with the control group, there were a significant decrease in BrdU/Nestin coexpression and remarkable decrease of p-Akt, p-CREB and BDNF mRNA and protein expression in LY294002group (P<0.05). However, there were a significant increase in BrdU/Nestin coexpression and remarkable increase of p-Akt, p-CREB and BDNF mRNA and protein expression in LY294002+Rolipram group (P<0.05).Conclusion:1. Intrauterine inflammation can cause white matter damage and induce learning and memory deficits in developing rats. Endogenous neurogenesis may occur in neonatal rats after intrauterine inflammation, and hippocampus neurogenesis may reverse or improve learning and memory deficits.2. There were significant increase of p-Akt, p-CREB and BDNF mRNA and protein expression in the E. coli infected group. Moreover, immunohistochemisty showed p-Akt,p-CREB and BDNF had expression in the DG of hippocampus. CREB signaling pathway may participate to regulate hippocampus neurogenesis.3. LY294002can decrease hippocampus neurogenesis, aggravate learning and memory deficits, and decrease p-Akt, p-CREB and BDNF mRNA and protein expression. However, the treatment of Rolipram could reverse or improve these impairments. Thus, we hypothesize that the possible Akt/CREB/BDNF signaling transduction pathway may be required for the regulation of neurogenesis and play an important role in improving learning and memory deficits.