Time-dependent effect of combination therapy with erythropoietin and granulocyte colony-stimulating factor in a mouse model of hypoxic-ischemic brain injury

Erythropoietin (EPO) and granulocyte colonystimulating factor (G-CSF) are likely to play broad roles in the brain. We investigated the effects of combination therapy with EPO and G-CSF in hypoxicischemic brain injury during the acute, subacute, and chronic phases. A total of 79 C57BL/6 mice with hypoxic-ischemic brain injury were randomly assigned acute (days 1–5), subacute (days 11–15) and chronic (days 28–32) groups. All of them were treated with G-CSF (250 μg/kg) and EPO (5 000 U/kg) or saline daily for 5 consecutive days. Behavioral assessments and immunohistochemistry for angiogenesis, neurogenesis, and astrogliosis were performed with an 8-week follow-up. Hypoxia-inducible factor-1 (HIF-1) was also measured by Western blot analysis. The results showed that the combination therapy with EPO and G-CSF in the acute phase significantly improved rotarod performance and forelimb-use symmetry compared to the other groups, while subacute EPO and G-CSF therapy exhibited a modest improvement compared with the chronic saline controls. The acute treatment significantly increased the density of CD31+ (PECAM-1) and α-smooth muscle actin+vessels in the frontal cortex and striatum, increased BrdU+/PSA-NCAM+neurogenesis in the subventricular zone, and decreased astroglial density in the striatum. Furthermore, acute treatment significantly increased the HIF-1 expression in the cytosol and nucleus, whereas chronic treatment did not change the HIF-1 expression, consistent with the behavioral outcomes. These results indicate that the induction of HIF-1 expression by combination therapy with EPO and G-CSF synergistically enhances not only behavioral function but also neurogenesis and angiogenesis while decreasing the astroglial response in a timedependent manner.