| Objective:This study established hypoxic ischemic brain damage model, given recombinant human granulocyte colony stimulating factor intervention, observation model group at different time points:rats sports behavior, brain morphology, brain cell apoptosis and detected the levels of TNF-a in blood, reveals the mechanism of G-CSF’s neuroprotective, open new way to clinical treatment of neonates hypoxic ischemic encephalopathy. Methods:one hundred twenty7days old SD rats, irrespective of gender, body weight12～18g were randomly divided into three groups:sham operation group, HIBD model group, rhG-CSF treatment group, each group40rats. HIBD model group and rhG-CSF treatment group were make HIBD model, each groups were divided into5subgroups according different kill times:6h group,24h group,48h group,72h group,7d group,each subgroup Contains8rats. Model group uses the way ligation the left common carotid artery then put rats in8%O2hypoxic environment2hours. The treatment group given rhG-CSF (60μg/kg) subcutaneous injection once a day for five days immediately after HIBD. observed the changes about sport behavior, brain morphology, HE staining, flow cytometry examination detected apoptosis in brain tissue, double ant body sandwich ELISA assay in blood TNF-a expression. Results:(1)neonatal rats after HIBD appear varying degrees of abnormal behavior.(2)Sham appearance of normal brain tissue, bilaterally symmetrical, without edema, atrophy and necrosis;HIBD model group at different time points after HIBD appear eye view different degrees changes of brain abnormality, but rhG-CSF treatment group at each time point the brain morphology was almost normal.(3)HE staining:sham operation group at different time points clear at the cellular level in brain tissue, cells with normal morphology; HIBD model group observed varying degrees of nerve cell swelling, degeneration after6h in HIBD, nerve cells of different degrees of necrosis, nuclear fragmentation, dissolution after72h in HIBD, necrosis is more evident at after7d in HIBD, the number of neurons significantly reduced, glial cell proliferation, cell derangement. rhG-CSF treatment group at each time point compared with HIBD model group was show Significantly Improve both in pathological changes in brain tissue and cell arrangement.(4) blood TNF-a expression:HIBD group, rhG-CSF group compared the control group at different time points of serum TNF-a significantly increased, there were significant differences (P<0.05). Sham group, HIBD group, rhG-CSF in serum TNF-a content in the6h were137.00±3.61ng/L、288.91±3.36ng/L、228.85±6.41ng/L、in the24h were139.02±1.59ng/L、303.59±3.32ng/L、215.23±7.01ng/L、in the48h were141.30±4.08ng/L、320.69±4.77ng/L、206.12±3.36ng/L、in the72h were140.30±4.36ng/L、336.80±3.10ng/L、197.19±6.55ng/L、in the7D were142.65±2.73ng/L、344.30±3.79ng/L、188.75±7.19ng/L、sham operation group at each time point, TNF-a values uniform and stable,HIBD group values of serum TNF-a gradually increased over time,72h to reach the peak, then gradually decreased with time, rhG-CSF group TNF-a. rhG-CSF serum levels of TNF-a values in the early to reduce gradually over time,but each time point compared to HIBD group show significantly difference (P <0.05), all time points rhG-CSF group compared with the sham group the value of TNF-a increased with significant differences (P<0.05).(5) flow cytometry rate of neuronal apoptosis:HIBD group, rhG-CSF group compared to control group at different time points the apoptosis in brain tissue was significantly higher, there were significant differences (P<0.05). Sham group, HIBD group, rhG-CSF group rate of apoptosis in brain tissue6h were4.35±0.32%、5.45±0.39%,4.86±0.38%;24h were5.13±0.97%13.05±1.6%、10.05±1.26%;48h were5.35±0.85%、13.29±1.51%10.58±0.99%;72h were5.53±0.94%、18.04±1.19%、12.05±0.66%;7d were4.62±0.69%、15.90±1.37%、6.03±1.23%; sham operation group the rate of brain cell apoptosis reach peaked at72h then gradually fall, in the HIBD group and rhG-CSF group also observed this trend, each corresponding time point HIBD group compared to rhG-CSF group have significant difference (P<0.05). Conclusions:(1)give rhG-CSF treatment to HIBD neonatal rats can reduce brain tissue of pathology and improve brain morphological changes in brain, suggesting that rhG-CSF have neuroprotective effects to HIBD.(2) HIBD model neonatal rats the value of serum TNF-a expression was significantly increased, after treatment with rhG-CSF serum TNF-a levels decreased significantly, suggesting that TNF-a Increase in hypoxic ischemic brain injury, serum TNF-a value significantly increased in the early Period (6hours), suggesting that monitoring TNF-a in serum can be used early prediction of brain injury and a good indicator observed treatment efficacy.(3) rhG-CSF treatment can significantly reduce the serum TNF-a expression of HIBD rat, suggesting that the neuroprotective mechanisms of rhG-CSF may by inhibiting expression of inflammatory factor TNF-a. |
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