| ObjectiveThis experiment produced the neonatal hypoxic-ischemic brain injury Neonate7-day-old SD rats rat model,and intranasal delivered insulin-like growth factor-1to central nervous system via olfactory,to search for the proliferation, migration and differentiation of endogenous neural stem cells by using immunohistochemical method,and to explor the nervous nutrition and protection of insulin-like growth factor-1; To investigate the changing of Foxgl gene in brain after HIBD by RT-PCR to explore the effect of IGF-1on the expression of Foxgl.Materials and Methods7-day-old SD rats were randomly divided into three groups:sham control group,model saline control group,and IGF-1-treated group. According to the sacrificed time point of rat after HIBD,each group were divided into five subgroups(1st,3th,5th,7th,14th day), there were a total15subgroups. The animal model of newborn rat HIBD was established using Rice method. Model saline control group was delivered by intranasal with0.1ml saline after HIBD1hour, IGF-1-treated group was delivered by intranasal with IGF-12.5ug(dissolved with0.1ml saline), and Meanwhile sham control group only split common carotid artery without deligation, nasal deliveryand hypoxia.Brdu, Nestin and NSE are respectly the marker of the new proliferating cell,neural stem cell and astroeytes. Brdu-Nestin marked the proliferated neural stem cells, Brdu-NSE signed the differentiated neural cells, and Brdu-GFAP labeled differentiated astrocyte cells.All animals were killed respectively on the1st,3th,5th,7th,14th day after hypoxia. Brdu was intraperitoneal injiected every four hours on the day before execution for a total of3times in a dose of50mg/kg in all animals which were going to be used for immunohistochemistry experiments.After four hours the brains were removed, fixed, sliced, HE staining were used to observe the pathomorphologic changes in injuryied brain; immunohistochemical staining method were used to detect and count the the numbers of BrdU positive cells, BrdU-Nestin double lable cells, BrdU-NSE double lable cells and BrdU-GFAP double lable cells in sub ventricular zone, hippocampus and cerebral cortex.The brains were normally removed which were used for RT-PCR. The expression of Foxgl genes was examined by RT-PCR.Results1Model identification:The behaval capability of all rats were normal before modeled. Nevertheless the rats can not stand up after molded, can not call and turn left when tail clamp Using HE staining to dispose paraffin section.In light microscope the neuron necrosis was observed in the left hemisphere,"red neurons" appeared in it. Pathological changes were distinctive in cerebral cortex and hippocampus.2Immunohistochemitry①Expression of Brdu positive cells and Brdu-Nestin double label positive cells: in model control group:the positive cells began to increase on1st day, reached the peak on3rd day, reduced on7th day and significantly reduced on14th day after HIBD. Brdu positive cells were,321.00±23.02,368.00±26.07,384.00±27.93,342.00±20.25,157.00±20.25; Brdu-Nestin-positive cells were10.00±2.28,24.00±5.66,21.00±3.41,15.00±3.41,8.00±2.83;in IGF-1-treated group:the positive cells began to increase on1st day, significantly increased on3rd day, reached the peak on5th day, lightly decreased on7th day, significantly reduced on14th day after HIBD. Brdu positive cells were357.00±11.40,438.00±20.25,476.00±27.39,385.00±11.83,204.00±22.80; Brdu-Nestin-positive cells were18.00±3.41,43.00±7.07,85.00±7.07,56.00±7.07,37.00±3.41; in sham operation group, there were no significant proliferation phenomenon. With the extension of time neural stem cells gradually returned to a resting state, Brdu positive cells were240.00±21.21,200.00±7.07,120.00±7.07,60.00±10.00; Brdu-Nestin positive cells did not express in brain. Compared with each other at the same point, the numbers of positive cells in IGF-1-treated group were higher than model control group than sham operation group, the differences were significant(P<0.05).②Expression of Brdu-NSE, Brdu-GFAP double label positive cells:in model control group, the positive cells were observed on7th day firstly, but the soma of the cell small with fine and short umbos and on14th day the cells were more, the soma of the cell was in star shape with thick and long umbos.growth, Brdu-NSE positive cells were15.00±2.27,25.00±3.96; Brdu-GFAP positive cells were35.00±3.96,45.00±4.72; in IGF-1-treated group, the positive cells appeared firstly on5th day,earlier than model control group. significantly increased on7th day in the brain, and reached its peak on14th day. Brdu-NSE positive cells were40.00±10.06,50.00±6.39; Brdu-GFAP were20.00±3.42,26.00±6.28. There were no expression in sham operation group. Compared between the two groups at same point, there was significant difference(P<0.05).3Results of RT-PCROn1st,3rd,7th,14thday,the expression of Foxgl was0.77±0.046,0.65±0.134,0.76±0.041,0.68±0.047,0.72±0.024in sham operation group, no significant change. In model control group:the expression of Foxgl reduced the first three days, then increased, and closed to the sham operation group on7th day, the results were0.60±0.037,0.53±0.056,0.62±0.054,0.74±0.053,0.78±0.043; In IGF-1-treated group:the trends of Foxgl expression was similar with model control group, but was higher than it, the difference was statistically significant(P<0.05), and the results were0.65±0.054,0.64±0.059,0.73±0.051,0.80±0.051,0.84±0.051.ConclusionThe nasal delivery of IGF-1can promote endogenous neural stem cell to proliferate, differentiate,after HIBD. IGF-1also contributes to the expression of Foxgl. |
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