| ObjectiveIschemia stroke is one of the commonest neurological diseases with relative high lethality and disabled rate.People have been trying to seek for efficient treatments for many years,but until now,there has been no breakthrough.Recently,a series of experiments suggested that ongoing neurogenesis in the dentate gyrus not only occurred in the normal adult mammalian brains,but also was enhanced by many physiological activities and pathological events.These findings provide possibility and feasibility for reconstructing cerebral infarct region,and will bring a new bright clue for treating cerebral vascular diseases.Basic fibroblast growth factor(bFGF) is a potent trophic factor for brain cells that distributes extensively among central nervous system and protects neurons against a number of toxins and insults.Evidence is emerging that the Wnt/β-catenin pathway may be important in regulating stem cell self-renewal and differentiation programs.Recent evidence suggests that the Wnt signaling pathway,along with its components,has been shown to be crucial in directing cell fate during embryogenesis.This pathway also regulates proliferation of primitive cells in adult tissues.To explore the mechanism of involvement of the Wnt/β-catenin pathway during regulated growth of neural stem cells in rats,we try to investigate the temporal profile of neurogenesis in the dentate gyrus after rat focal ischemic middle cerebral artery occlusion/reperfusion and to explore the possible mechanisms related to neurogenesis by systemic BrdU to label dividing cell. we investigated the regularity of neurogenesis in the dividing cells.Firstly,we investigated the regularity of neurogenesis in the dentate gyrus after focal ischemic after reperfusion 3d,1w,2w,3w.Secondly,we observed the effects of Wnt signals molecule on neurogenesis in the dentate gyrus after focal cerebral ischemia,and discussed the role of Wnt signals molecule enhanced neurogenesis.Lastly,we investigated the influence of bFGF on Wnt signals molecule in the hippocampus after focal cerebral ischemia reperfusion.Levels of Wnt1,β-catenin during rat endogenous neural stem cell regeneration use reverse transcriptase-polymerase chain reaction (RT-PCR) and Western-blot and immunohistochemistry techniques.Materials and Methods1,Middle cerebral artery occlusion(MCAO) by sulture emboli was established with health male rats.Sham-operation contral rats received the operations without ischemia.Horner's sign in left side and paralysis of the right front limbs were observed to judge the success of MCAO model.2,After focal cerebral ischemia we observe morphological chang in the hippocampus and cortex by HE and electron microscope.3,C-Myc expression and neuron apoptosis were detected in the cortex and hippocampus by Immunohistochemisty and TUNEL after focal cerebral ischemia,and intervention of bFGF.4,The regularity of proliferation of NSC were detected at different time by BrdU labelling and Immunohistochemisty after focal cerebral ischemia.5,The Wnt-1 andβ-catenin expression were detected in the hippocampus by Western blot analysis.6,After focal cerebral ischemia,The Wnt-1 andβ-catenin mRNA expression were detected in the hippocampus by RT-PCR.7,Theβ-catenin expression were detected in the cortex and hippocampus by Immunohistochemisty,and intervention of bFGF. 8,Theβ-catenin expression were detected in the cortex and hippocampus by in situ hybridization,and intervention of bFGF.Result1,Middle cerebral artery occlusion(MCAO) by sulture emboli was established with health male rats.Sham-operation contral rats received the operations without ischemia.Horner's sign in left side and paralysis of the right front limbs were observed to judge the success of MCAO model.2,HE and electron microscope result:In the sham-operation group,there were no obvious morphological changes in cerebral cortex and hippocampal neurons of rats; In the model group,the morphology of neuron is damaged.But the bFGF can improve this kind of damage.3,TUNEL result:No apoptotic cells were observed in sham-operation group.In ischemia-reperfusion group apoptotic neurons in ischemic region of cortex and hippocampus enhanced.The decrease of apoptotic neurons in ischemic region of cortex and hippocampus were seen in bFGF treatment group.Compared with ischemia-reperfusion group,the diference was significant.All of above indicated that apoptosis existed in ischemic region of cortex and hippocampus and bFGF could reduce neuronal apoptosis after cerebral ischemia reperfusion.4,Result of immunohistochemisty indicated c-Myc expression in the cortex and hippocampus tissue of rats was at low level in the sham-operation group,and was significantly increased in the model group;The c-Myc expression in the cortex and hippocampus tissue of rats was markably decreased in the treatment group than in the model group.5,Result of immunohistochemisty indicated most BrdU positive cells located in the DG.BrdU positive cells reached maximally at 7d after the cerebral ischemia, compared with the the sham-operation group,bFGF can increase the BrdU positive cells.6,Result of Western Blot indicated that no detectable levels of Wnt1 were found in the hippocampus,Level ofβ-catenin mRNA was shown to be low.Level of Wnt1 andβ-catenin increased at this time after 3d cerebral ischemia.Levels of Wnt1 andβ-catenin reached maximally at 7d and 14d respectively.At 21d decreased levels of Wnt1 andβ-catenin were observed.Level of Wnt1 andβ-catenin decreased to less than normal level.The result of RT-PCR similar with Western Blot.7,Result of immunohistochemisty indicated that Theβ-catenin expression in the cortex and hippocampus tissue of rats was at low level in the sham-operation group, and was significantly increased in the 14d model group;Theβ-catenin expression in the cortex and hippocampus tissue of rats was markably increased in the treatment group than in the model group.The result of in situ hybridization is similar with immunohistochemistyConclusions1,bFGF suppresses neuronal apoptosis,inhibit c-Myc expression,therefore protect brain tissue when cerebral ischemia reperfusion.2,After focal cerebral ischemia we detect the regularity of proliferation of NSC for different time by BrdU labelling.Proliferation of endogenous neural stem cells was stimulated by focal cerebral ischemia,bFGF promote neural stem cells proliferation.3,Explore the mechanism of involvement of the Wnt/β-catenin pathway during regulated growth of neural stem cells in rats.bFGF promote it expression.Our findings indicate that the Wnt/β-catenin pathway plays a role in proliferation and differentiation of neural stem cells.
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