| Background: Hypoxic-ischemic brain damage (HIBD) is a common disease in neonate. It is a very important risky factor which causes cerebral palsy and harms children's health. In the 2nd national investigation, the ratio of unfavourable prognosis was still as high as 20.8% in 2003, so the therapeutic methods of HIBD are limited. In recent years, many researchers found that there were neural stem cells (NSC) in the brain of newborn animals. Endogenous NSC can proliferate reactively and differentiate to repair the structure and function of brain after HIBD. But some researchers found that the ability of proliferation and differentiation of NSC was limited and the ratio of differentiation is low. Using 5-bromo-2-deoxyuridine (BrdU) marker to observe the differentiation of NSC in hippocampus of newborn rats who suffered from HIBD, Liu and his colleagues found that NSC couldn't differentiate into mature neurons in 15 days, and after 26 days only 27% cells marked by BrdU could differentiate into mature neurons. Masanori and his colleagues found that the quantity of NSC increased 3 fold in subependymal region within 10 days in newborn rats who suffered from HIBD, but after 60 days only a few cells could differentiat into astrocyte which express glial fibrillary acidic protein (GFAP) and neurons which express neuron-specific nuclear antigen (NeuN). How to improve the proliferation and differentiation ratio of endogenous NSC needs to be studied. Recently researchers found regional microenvironment could affect the differentiation of NSC in brain. Whether it played a key role in inhibiting the proliferation and differentiation of endogenous NSC, or in causing the low ratio of differentiation of endogenous NSC and leading NSC failed in rebuilding the structure and function of brain effectively? All of these need to be studied. If we can find this key factor and use more effective therapeutic methods, the prognosis of HIBD will be improved greatly.Forkhead box G1(Foxg1) gene is a very important member of Forkhead-box(FOX)gene family, researchers has found that Foxg1 is a key gene which controls the proliferation and differentiation of NSC, and it will cause dysdifferentiation of NSC and brain dysplasia if the express of Foxg1 is blocked. The expression of Foxg1 was persistence in postnatal animals'brain (Cerebral cortex, hippocampus, olfactory bulb, basal ganglia, et al ). These regions were also damaged easily, when HIBD happened. Many studies found that after HIBD the rebuilding of brain structure and function like embryonic brain development, Foxg1 may play a important role in the differentiation and proliferation of NSC after HIBD. Objective: To investigate the changing of Foxg1 gene and other specified genes which expressed in cerebral cortex, including Foxp2, ER81, Otx1, Foxp1, ROR? and Reelin. To investigate the changing of Cajal-Retzius cells after HIBD, and to explore the relationship between Cajal-Retzius cells and Foxg1.Methods: The animal model of newborn rat HIBD was established using Rice method. On the 3rd 7th 14th 28th and 56th day, the right cerebral hemisphere was resected and preserved. The expression of each genes was examined by RT-PCR, and the quantity of Cajal-Retzius cell was examined by immunohistochemistry.Results: 1) Model identification: Using HE staining to dispose paraffin section. In light microscope the neuron necrosis which characterized as vacuole, karyopyknosis, faint structure of tissue and inflammatory cells proliferation was observed in the right hemisphere. Pathological changes were distinctive in cerebral cortex and hippocampus. 2) Results of RT-PCR: Foxg1 On 3rd , 7th , 14th , 28th , 56th day, the expression of Foxg1 is 0.524±0.086, 0.726±0.067, 0.770±0.122, 0.786±0.083, 0.882±0.081 respectively in experiment group, and 0.798±0.121, 0.787±0.078, 0.664±0.109, 0.744±0.096, 0.757±0.065 respectively in control group. Compared to control group , the difference is significant in the 3rd and 56th day groups. Generally, the expression of the Foxg1 is down-regulated in the first 7 days in experimental group, while Foxg1 expressed steadily in control group. Reelin On 3rd , 7th ,14th , 28th , 56th day, the expression of Reelin is 0.591±0.034, 0.542±0.131, 0.533±0.085, 0.694±0.063, 0.668±0.144 respectively in experiment group, and 0.565±0.113, 0.329±0.062, 0.474±0.081, 0.553±0.073, 0.618±0.104 respectively in control group. Compared to control group, the difference is significant in the 7th and 28th day. Generally, in experimental group the expression of the Reelin is up-regulated on every time point. Foxp1 On 3rd , 7th , 14th , 28th , 56th day, the expression of Foxp1 is 0.616±0.056, 0.886±0.096, 0.710±0.059, 0.636±0.070, 0.698±0.109 respectively in experiment group, and 737±0.115, 0.743±0.102, 0.746±0.108, 0.735±0.082, 0.899±0.064 respectively in control group. Compared to control group, the difference is significant in the 3rd , 7th , 28th and 56th day. Generally, the expression of the Foxp1 is down-regulated in experimental group. ER81 On 3rd , 7th , 14th , 28th , 56th day, the expression of ER81 is 0.654±0.096, 0.942±0.068, 0.724±0.094, 0.838±0.105, 0.977±0.123 respectively in experiment group, and 0.710±0.120, 0.666±0.139, 0.797±0.107, 0.941±0.141, 1.027±0.113 respectively in control group. Compared to control group, the difference is significant in the 7th day. Generally, the expression of the ER81 is down-regulated in experimental group. Otx-1 On 3rd day we didn't detect the expression of Otx-1 in experiment group, but we could detect it in control group. After 7th day we could detect the expression of Otx-1 in experiment and control group. On 3rd , 7th , 14th ,28th , 56th day, the expression of Otx-1 is 0.000±0.000, 0.713±0.122, 0.570±0.134, 0.535±0.037, 0.551±0.044 respectively in experiment group, and 0.571±0.098, 0.466±0.137, 0.463±0.133, 0.462±0.121, 0.401±0.061 respectively in control group. Compared to control group, the difference is significant in the 7th and 56th day. RORβOn 3rd , 7th ,14th , 28th , 56th day, the expression of RORβis 00.951±0.118, 0.839±0.078, 0.786±0.114, 0.828±0.109, 1.120±0.204 respectively in experiment group, and 00.984±0.133, 0.868±0.169, 1.060±0.225, 1.238±0.155, 0.854±0.094 respectively in control group. Compared to control group, the difference is significant in the 14th , 28th and 56th day. Generally, in experimental group the expression of the RORβis down-regulated in the early stage of HIBD. Foxp2 On 3rd , 7th ,14th , 28th , 56th day, the expression of Foxp2 is 0.460±0.107, 0.526±0.119, 0.566±0.164, 0.455±0.050, 0.570±0.061 respectively in experiment group, and 0.543±0.104, 0.420±0.163, 0.452±0.055, 0.469±0.043, 0.422±0.055 respectively in control group. Compared to control group, the difference is significant in the 14th and 56th day. Generally, in experimental group the expression of the Foxp2 is down-regulated in the early stage of HIBD. 3) Results of immunohistochemistry The results of immunohistochemistry showed that the Cajal-Retzius cells were proliferated, and have polymerization tendency. On 3rd , 7th ,14th , 28th , 56th day , the positive cells were 24.9±2.424, 31.4±4.452, 42.3±2.908, 47.1±1.853, 45.5±2.173 respectively in experiment group, and 21.7±2.627, 21.7±3.199 , 21.8±3.190 , 22.4±2.221,27±2.000 respectively in control group. Compared to control groups, the difference is significant in all observing days. 4) statistics results In the normal conditions (control group) the relation between Otx1 and Foxg1 is related, but in experiment group the relation actually turned inverse correlation. The relation between Reelin and Foxg1 presents inverse correlation in control group, but in experiment group the relation actually turned related. The relations between Foxg1 and other special genes (Foxp1, ER81, RORβ, Foxp2) present inverse correlation in both control and experiment group.Conclusions: After HIBD, the expression of Foxg1 in newborn rat is down-regulated, Reelin is up-regulated while the other specified genes of cerebral cortex(Foxp2, ER81, Otx1, Foxp1, ROR?) are down-regulated. In early stage of HIBD, many endogenesis NSC differentiate to Cajal-Retzius cells because of the down-regulated of Foxg1 and endogenesis NSC differentiating to mature neurons in cerebral cortex reduced correspondingly. So the early rebuilding of brain after HIBD is inhibited. After HIBD, the relation between Foxg1 and Reelin is related, and the relation between Foxg1 and Otx-1 presents inverse correlation. The relations between Foxg1 and other special genes (Foxp1, ER81, RORβ, Foxp2) are not obvious. Whether Foxg1 control the expression of specified genes directly still need further studies.
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