The Effects Of G-CSF On The Anti-inflammation Of Hippocampal Microglial Cells And Functional Protection In Hypoxic Ischemic Neonatal Rats

Background:Neonatal Hypoxic Ischemic Brain Damage(HIBD)is an event of hypoxia and asphyxia during perinatal period in neonates,which is a common and high risk of clinical disease,with quite a few complications and sequelae.Clinical treatment such as hypothermia and hyperbaric oxygen has limited effects and the prognosis is far from satisfactory,with the addition of tissue damage,infection or other adverse effects.Granulocyte colony stimulating factor(G-CSF)is a natural glycoprotein molecules which can be purified from human body.It is produced by various tissues including mononuclear macrophages,endothelial cells,fibroblasts and mesothelial cells,etc.G-CSF has profound and pleiotropic effects on proliferation,differentiation and maturation of neutrophils.Besides it can be used for cell mobilization,anti-inflammation and anti-apoptosis.Herein,the aim of this study was to evaluate the anti-inflammatory effects of G-CSF on hippocampal microglial cells after hypoxic and ischemic damage to newborn rats,and to evaluate its long-term neuroprotective function.In order to provide a reference for clinical drug therapy.Objective:To observe the effect of granulocyte colony-stimulating factor on the anti-inflammatory protection of hippocampal microglia cells in hypoxic ischemic rat pups,and the neuroprotection of learning and memory function related to hippocampus.Methods:There are two parts in this study.Part 1 includes a total of 90 newborn SD rats at age of 7 days.They are randomly divided into control group(SHAM),injury group(HIBD)and treated group(G-CSF),with 30 rats in each group.Each group is further divided into 5 subgroups according to 5 time points,that is,3 days after damage(D3),5 days after damage(D5),7 days after damage(D7),14 days after damage(D14)and 21 days after damage(D21),with 6 pups in each subgroup.HIBD group and G-CSF group are treated with ligation of right common carotid artery and hypoxia.SHAM group is only exposed to surgery with neither ligation nor hypoxia.In addition,G-CSF group received intraperitoneal injections of 60 μg/kg G-CSF at 1 hour after injury,1 day after injury and 2 days after injury.HIBD group and SHAM group were both injected with same amount of normal saline.The 3 groups were sacrificed on D3,D5,D7,D14 and D21 after injury.They were operated with cardiac perfusion.Brain tissues were collected to measure the coexpression of microglial and inflammatory cytokines at D3,D5 and D7 post-injury,and compare the expression levels of pro-inflammatory and anti-inflammatory cytokines at different time points between groups.In part 2,we randomly divided 30 SD rat pups aged 7 days into three groups,SHAM group,HIBD group and G-CSF group,each of 10 rats.The modeling approach and drug therapy kept the same with part 1.Morris water maze behavioral test was performed at the age of 2 months.Results:Experimental results in part 1 showed significantly increased number of microglia cells on D3 after hypoxic ischemic injury,followed with decreasing number on D7 and D14.After damage,the co-expression cells of both Iba-1/TNF-a and Iba-1/TGF-p increased.Compared with HIBD group,co-expression of Iba-1/TGF-βin G-CSF group was higher,and the co-expression peak of Iba-1/TNF-a was delayed.The fluorescence positive samples of TNF-α reached to peak on 3 days after damage,then gradually decreased and stabilized.After G-CSF treatment,TNF-a level was significantly lower than the injury group.The fluorescence samples of TGF-βwere higher on D3 and D5 after injury,G-CSF could increase the expression amount and prolong the peak time of TGF-β.Results in part 2 indicated that time for the rat to find platform shortened with increasing training days.In the fourth training day,spending time of HIBD group was significantly higher than that of SHAM group.While the navigation time of G-CSF group was shorter than that of HIBD group,there was no statistical difference.In the spatial probe test,the residence time of HIBD group spent in the target quadrant and the frequency of platform crossings were significantly lower than SHAM group.Although the residence time and platform crossings of G-CSF group were more than HIBD group,the difference was not significant.Conclusion:G-CSF can promote the activation and expression of microglial cells in the hippocampal dentate gyrus after hypoxic ischemic injury,it has obvious anti-inflammatory protection effect and improves the long-term learning and memory function of hippocampus.