The Effect Of Granulocyte Colony-stimulating Factor On Neurogenesis And Angiogenesis In Vivo After Hypoxic-ischemic Brain Damage In Rats

BackgroundHypoxic-ischemic brain damage(HIBD)is a disease in neonates that affects the energy metabolism of brain cells,causing apoptosis and necrosis,which in turn cause irreversible damage to the brain,and result in disability and death.Therefore,taking measures to cut off the pathological process and reduce the long term sequelae is important.At present,neonatal HIBD still lacks specific therapies,and mild hypothermia therapy is considered feasible.However,because of the time-window,there is no obvious curative effect in the subacute and chronic phase of HIBD;hyperbaric oxygenation can effectively increase brain oxygenation and reduce brain damage.However,due to the restriction of medical condition,fewer and fewer hospitals can adopt systematic and standardized treatment.Stem cell transplantation has been proved to be an effective treatment method in animal experiments,which can improve its motor development and cognitive function.Stem cell-associated factors such as Erythropoietin,and Granulocyte colony-stimulating factor(G-CSF)are currently being studied.G-CSF is a growth factor that stimulates the proliferation and differentiation of myeloid hematopoietic cells.After binding with receptors,it activates a variety of signal transduction pathways,then activates downstream substrates,which is affecting cell proliferation and differentiation,and exerting protective effects such as inducing neurogenesis and angiogenesis.ObjectiveTo explore the effect and possible mechanism of granulocyte colony-stimulating factor on neurogenesis and angiogenesis in vivo after hypoxic-ischemic brain damage in neonatal Sprague Dawley(SD)rats.MethodWe chose 126 neonatal 7-day-old SD rats which were randomly assigned into 3 groups:sham operation group,model group and G-CSF group.According to the modified method of Rice model,we builded the hypoxic-ischemic brain injury model in the model and G-CSF group.The sham operation group only dissected the right common carotid artery without ligation and hypoxia.The G-CSF group was intraperitoneally injected with G-CSF every day for 7 days after the model was established,and the dose is 50 ?g/kg.And then we recorded the eye-opening time in each group and monitored the body weight of each group every 3 days;The neurobehavioral scores of rats in each group at P21 and P28 after building the model;At 14,21 and 28-day-old rats in each group,pathological changes in the subventricular zone and hippocampal dentate gyrus were observed by hematoxylin-eosin(HE)staining.The expression of vascular endothelial growth factor(VEGF)in the subventricular zone and dentate gyrus of the hippocampus was detected by immunohistochemistry.immunofluorescence staining of BrdU and BrdU/DCX was detected the dynamic changes of neuronal proliferation in subventricular zone and dentate gyrus of hippocampus.Western blot was used to detect the expression of GSK3? and p-GSK3? in right brain of rats in each group at P14.Result1.Growth development and neurobehavioral scores:Rats in the G-CSF group had shorter eye-opening time than the model group;Body weight of the rats in each group increased as the number of days increased,and the body weight of the G-CSF group was significantly higher than that of the model group at P28;The Garcia scores of the G-CSF group were improved compared with the model group at P21and P28,P<0.05.2.HE staining:Compared with the model group,at 14,21 and 28-day-old,the G-CSF group had improved pathological morphology in the subventricular zone and hippocampal dentate gyrus,the neuronal arrangement was close and orderly,and the new neurons increased.3.Immunohistochemistry of VEGF:Compared with the model group,the intensity of VEGF expression in the dentate gyrus of the hippocampus in the G-CSF group were all significantly higher at P14,P21,and P28,P<0.05;In subventricular zone,the expression intensity of VEGF in 28-day-old rats in the G-CSF group was significantly higher than that in the model group,P<0.05.4.BrdU/DCX double immunofluorescence labeling:BrdU and BrdU/DCX double-labeled positive cells in the subventricular zone and the dentate gyrus of hippocampus in each group showed a decreasing trend with the increase of the number of days.Compared with the model group,the number of BrdU and BrdU/DCX double-labeled positive cells in the dentate gyrus of the hippocampus increased significantly in the G-CSF group at P14 and P21,whereas in the subventricular zone,in the G-CSF group,BrdU and BrdU/DCX double-label positive cells significantly increased than the model group at P14,P21and P28,P?0.05.5.The expression of GSK3? and p-GSK3?:There was no significant difference in the expression of GSK3? among the groups.However,compared with the model group,the ratio of p-GSK3?(Ser9)to GSK3? expression was significantly lower in the G-CSF group,P<0.05.ConclusionG-CSF could promote neurovascular regeneration,improve the growth development and neurobehavioral scores after hypoxic-ischemic brain damage in rats.And G-CSF could down-regulate p-GSK3? and then increase GSK3? activity.