The Effect Of Granulocyte Colony-Stimulating Factor Carried By Fibrin Gel On Promoting Neovascularization In Rat Hindlimb Ischemia Model

Objective: To study the effect of G-CSF carried by fibrin gel on the total number ofperipheral white blood cells and neovascularization of ischemic muscle in rathindlimb ischemia model.Materials and methods:1, Then fibrin gel were made by mixing sealer protein and catalyst containingG-CSF, then added with PBS and incubated at37℃. At24,48,72, and96hours, thesupernatant was collected, the amounts of G-CSF in the supernatants were determinedwith ELISA method and the cumulative release rate of G-CSF was calculated.2, Thirty male SD rats were subjected to right hindlimb ischemia and randomlydivided into three groups: Gel+G-CSF, G-CSF, and PBS, and were respectivelyinjected with Gel+G-CSF, G-CSF and PBS. Before operation and at postoperativedays1,2,3,5and7, WBCs number was counted. The first and4weeks after surgery,5rats in each group were sacrificed. Muscle samples were harvested. Frozen sectionswere histochemically stained for alkaline phosphatase to detect capillary and countcapillary density, stained with hematoxylin to detect infiltrated cells, andimmunohistochemically stained for α-smooth muscle actin and VEGF to detectα-SMA positive vessel and expression of VEGF, count α-SMA positive vesseldensity.Results:1, Release of Gel+G-CSF in vitro: the cumulative release rate of Gel+G-CSFwere52.5±4.50%,76.3±4.47%and87.9±4.38%respectively at24,48and72hours.It seems that most of G-CSF was gradually released from Gel+G-CSF within threedays.2, WBCs counts of peripheral blood: The peak value of WBCs counts in groupGel+G-CSF (14.69±1.11×109/L) appeared at postoperative day1was significantlylower than that in group G-CSF (21.00±2.26×109/L) appeared at postoperativeday3. 3, Neovascularizaton effect: The capillary density in group Gel+G-CSF (686±108/mm2) was significantly higher than that in group G-CSF (491±110/mm2) orgroup PBS (252±78/mm2), P <0.05. The α-SMA positive vessel density in groupGel+G-CSF (6.1±0.8/mm2) was significantly higher than that in group PBS (2.6±1.3/mm2), P <0.05.4, Cell infiltration and expression of VEGF: there were more infiltrating cellsand expression of VEGF in ischemic tissue in group Gel+G-CSF than that in groupG-CSF or group PBS.Conclusion: Gel+G-CSF could promote the neovascularizaton in rat ischemichindlimb by promoting cell infiltrating into ischemic muscle, and activating thesecells to secrete VEGF. Intramuscular injection with Gel+G-CSF had stronger effecton nevascularization and more modest effect on WBCs counts than intramuscularinjection with G-CSF.