| Objective: Neonatal hypoxic-ischemic encephalopathy (HIE) is a common neonatal disease, it can cause neonatal death and permanent nervous system injury after neonatal period, and there are no effective therapeutic methods at present. Our study established hypoxic-ischemic brain damage (HIBD) model and gave recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment to observe the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor -2 (VEGFR2) in brain tissue at different time points, thereby to reveal the possible mechanism of neuroprotection of G-CSF and develop new method for the therapy of HIE.Methods: A total of 168 female or male Wistar rats aged 7d, with the birth weight of 12~18 gram were randomly divided into three groups: sham operation group, HIBD model group, rhG-CSF treatment group (n=56 in each group), HIBD model of neonatal rats were established in HIBD model group and rhG-CSF treatment group. Then each group was randomly divided into seven subgroups (n=8 in each subgroup) based on different time points at 6h, 12h, 24h, 48h, 72h, 5d, 7d after HIBD. HIBD model rats were prepared by keeping rats into hypoxic environment of 8% O2 concentration for 2 hours after clamping left common carotid artery. Rats in rhG-CSF treatment group were given rhG-CSF treatment (60ug/kg body weight, subcutaneous) instantly after HIBD, and were given on four subsequent days (five total injections). Rats in three groups were sacrificed at different time points and brain tissue were collected. Then morphologic changes of brain tissue in general obtained through macroscopic observation, the pathological changes of left brain tissue were observed under light microscope, and the expressions of VEGF and VEGFR2 in left brain tissue were detected with immunohistochemistry.Results: (1) Neonatal rats had various abnormal behaviors after HIBD. (2) Various morphologic abnormality of brain tissue of rats in HIBD model group at different time points were observed, but there were no obvious changes of morphology of brain tissue of rats in rhG-CSF treatment group. (3) HE staining: Cellular layer was distinct, cellular morphous was normal and there were no obvious never cell loss in brain tissue in sham operation group. Different extent cellular swelling and degeneration were found at 6h, 12h after HIBD in HIBD model group, cellular necrosis became apparent at 24h, 48h: cellular nuclear fragmentation, solution, cellular necrosis was more significant at 72h, at 5d, 7d neuron loss, gliocyte proliferation were observed, and cell layers were indiscriminate. In rhG-CSF treatment group, the pathological changes of brain tissue lessened significantly compared with HIBD model group, and cell arranged regularly. (4) Expressions of VEGF and VEGFR2: Both VEGF and VEGFR2 positive staining showed buffy fine grain deposition, positive cells were found in cerebral cortex and hippocampus, and positive staining was mainly in cytoplasm and axon process of nerve cell. There were no obvious VEGF and VEGFR2 positive cell in brain tissue in sham operation group at each time point; VEGF and VEGFR2 positive cells increased gradually in brain tissue in HIBD model group after HIBD, the peak time of positive expression was 24-48h after HIBD, afterward positive expressions decreased gradually, differences of VEGF, VEGFR2 positive expressions at different time point in HIBD model group had statistical significance (P<0.01), and the expressions of VEGF, VEGFR2 positive cell were increased significantly compared with sham operation group at each time point (P<0.01); VEGF, VEGFR2 positive cells were obvious in brain tissue in rhG-CSF treatment group at each time point, the expressions of VEGF, VEGFR2 positive cells were increased significantly compared with HIBD model group and sham operation group respectively at the each time point (P<0.01). (5) The expressions of VEGF and VEGFR2 in brain tissue after HIBD had notable positive correlation (r=0.778, P<0.01).Conclusions: (1) RhG-CSF treatment after HIBD in neonatal rats can reduce pathological changes of brain tissue, which suggests that rhG-CSF has neuroprotective effect. (2) The expressions of VEGF and VEGFR2 increased in brain tissue of neonatal rats after HIBD, which suggests the two factors has some protective effect in the morning period of HIBD. (3) RhG-CSF treatment can increase the expressions of VEGF and VEGFR2 in brain tissue of neonatal rats after HIBD, which maybe one of the neuroprotective mechanism of rhG-CSF.
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