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Phase-dependent effect of the combination therapy of erythropoietin and granulocyte-colony stimulating factor on hypoxic-ischemic brain injury

Other Titles
 허혈성 뇌손상에서 erythropoietin과 granulocyte-colony stimulating factor의 병합투여의 시기에 따른 효과 
Issue Date
Dept. of Medical Science/석사
Erythropoietin (EPO) is the major growth regulator of the erythroid cell lineage. EPO is to regulate the proliferation, maturation, and survival of erythroid progenitor cells in human bone marrow, in an oxygen-dependent manner. Therefore, EPO is a promising candidate drug for the treatment of various neurological diseases. Especially, the beneficial effects of EPO and its variants in models of experimental stroke and cerebral hypoxic–ischemic can be attributed to a multitude of cytoprotective mechanisms, including inhibition of apoptosis, anti-inflammatory, anti-oxidant actions and restoration of blood-brain barrier integrity, as well as stimulation of neurogenesis and angiogenesis. Granulocyte-colony stimulating factor (G-CSF) was previously identified among a set of humoral factors on which the survival, proliferation, and differentiation of hematopoietic cells in cell culture assays were dependent. G-CSF was also shown to exhibit several beneficial effects in animals with stroke. In particular, G-CSF improves functional recovery after stroke even when treatment is initiated at delayed time intervals. A recent study reported that G-CSF upregulates EPO expression via the induction of hypoxia-inducible factor-1α (HIF-1α) activity and that the combination therapy of EPO and G-CSF synergistically enhances neural survival and angiogenesis in animal models of cerebral ischemia. This study aimed to investigate via an animal model the effect of the combination therapy of EPO and G-CSF on hypoxic-ischemic brain injury treated during the acute, subacute, and chronic phases.Brain damage was induced in 12-weeks old C57BL/6 mice by unilateral carotid artery ligation and exposure to hypoxia condition (8% O2 for 45 min). After inducing hypoxic-ischemic brain injury, mice were randomly assigned to the acute phase (days 1 to 5), subacute phase (days 11 to 15) or chronic phase (day 28 to 32), study groups. All of which were treated with 250 μg/kg of recombinant human G-CSF and 5000 unit/kg of recombinant human EPO dissolved in saline solution once per day for 5 consecutive days. As a respective control group, they were treated with saline solution injection. Rotarod, forelimb-use asymmetry, and grip strength tests were performed to evaluate behavioral function. HIF-1α protein was measured by western blot. Angiogenesis, neurogenesis and glial scar marker were measured via immunohistochemistry. The combination therapy of EPO and G-CSF in acute phase treatment group significantly improved rotarod latency time, forelimb-use asymmetry, and grip strength compared to those of the other groups.The combination therapy during acute phase increased BrdU+/PSA-NCAM+ neuronal numbers and CD31+ vessels density, but GFAP+ astrocytic density significant decreased at post-treatment 2 weeks. CD31+ and α-SMA+ vessels density significant increased at post-treatment 8weeks. Furthermore, the treatment during acute phase significant increased the HIF-1α expression in cytosol and nucleus, whereas the chronic phase did not change the HIF-1α expression. This investigation demonstrated that induction of HIF-1α expression through the combination therapy of EPO and G-CSF during acute phase synergistically enhances not only motor performance but also neurogenesis and angiogenesis in hypoxic-ischemic brain injury. Especially, overexpression of HIF-1α in both cytosol and nucleus demonstrated a similar pattern with functional outcome, suggesting HIF-1α was related with the functional outcome. Therefore, the combination therapy of EPO and G-CSF may more contribute to functional recovery in the phase-dependent manner in hypoxic-ischemic brain injury.
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