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

Erythropoietin （EPO） and granulocyte colony- stimulating 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 hypoxic- ischemic 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, neuro- genesis, 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 a-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 time- dependent manner.