Extension of Tissue Plasminogen Activator Treatment Window by Granulocyte-Colony Stimulating Factor in a Thromboembolic Rat Model of Stroke

Abstract

When given beyond 4.5 h of stroke onset, tissue plasminogen activator (tPA) induces deleterious side effects in the ischemic brain, notably, hemorrhagic transformation (HT). We examined the efficacy of granulocyte-colony stimulating factor (G-CSF) in reducing delayed tPA-induced HT, cerebral infarction, and neurological deficits in a thromboembolic (TE) stroke model, and whether the effects of G-CSF were sustained for longer periods of recovery. After stroke induction, rats were given intravenous saline (control), tPA (10 mg/kg), or G-CSF (300 μg/kg) + tPA 6 h after stroke. We found that G-CSF reduced delayed tPA-associated HT by 47%, decreased infarct volumes by 33%, and improved motor and neurological deficits by 15% and 25%, respectively. It also prevented delayed tPA treatment-induced mortality by 46%. Immunohistochemistry showed 1.5- and 1.8-fold enrichment of the endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 in the ischemic cortex and striatum, respectively, and 1.7- and 2.8-fold increases in the expression of the vasculogenesis marker von Willebrand factor (vWF) in the ischemic cortex and striatum, respectively, in G-CSF-treated rats compared with tPA-treated animals. Flow cytometry revealed increased mobilization of CD34+ cells in the peripheral blood of rats given G-CSF. These results corroborate the efficacy of G-CSF in enhancing the therapeutic time window of tPA for stroke treatment via EPC mobilization and enhancement of vasculogenesis.

Keywords: G-CSF; hemorrhagic transformation; tPA; thromboembolic model; vasculogenesis.

Figure 1

Effects of granulocyte-colony stimulating factor (G-CSF) on delayed tissue plasminogen activator (tPA)-induced hemorrhage and cerebral infarction in rats subjected to thromboembolic stroke. (A) Photographs are representative coronal brain sections obtained 3 days after stroke, showing that tPA treatment produced remarkable hemispheric hemorrhage, whereas the combined treatment with G-CSF did not result in bleeding; (B) Infarct volume was reduced, and hemorrhage was not observed in rats treated with G-CSF 7 days after stroke; (C) Representative coronal brain sections stained with 2,3,5-triphenyltetrazolium chloride (TTC) 3 days after stroke showing the infarct area (white) and intact areas (red); (D) Quantitative analysis of spectrophotometric assay findings showing the incidence of hemorrhage in rats subjected to delayed tPA treatment, which was reduced by 47% by G-CSF treatment; (E) Quantitative analysis of infarct volume in vehicle-, tPA-, and G-CSF + tPA-treated groups. G-CSF reduced infarct volume by 33% in stroked rats subjected to delayed tPA treatment; * p < 0.05, ** p < 0.01, *** p < 0.001, n = 7 rats per group. The data are expressed as mean ± S.E.M.

Figure 2

Effects of G-CSF on delayed tPA-induced motor and neurological deficits and mortality. (A) G-CSF treatment reduced delayed tPA-induced motor deficits by 15% as measured by the elevated body swing test (EBST); (B) G-CSF also reduced stroke and/or delayed tPA-induced neurological impairment by 25% when administered 7 days after stroke; (C) G-CSF decreased the incidence of mortality by 46% in stroke rats given tPA 6 h after stroke; ** p < 0.01 tPA vs. G-CSF + tPA, n = 10 rats per group. The data are expressed as mean ± S.E.M.

Figure 3

Immunohistochemical analyses of endothelial progenitor cell (EPC) markers CD34+ and VEGFR2 and of the vasculogenesis marker vWF in the ischemic cortex and striatum. Representative merged images obtained 3 days after stroke show co-localization of CD34+ and VEGFR2 or vWF with 4′,6-diamidino-2-phenylindole (DAPI; blue filter, nuclear staining). Analysis of fluorescence intensities showed that G-CSF treatment increased the expression of CD34+ and VEGFR2 in the ischemic cortex by 1.8- and 1.5-fold (vs. control) 3 and 7 days, respectively, after stroke. G-CSF also increased CD34+ and VEGFR2 expression in the ischemic striatum by 2.27- and 1.8-fold (vs. control) 3 and 7 days, respectively, after stroke. vWF expression was markedly increased by 2- and 1.7-fold (vs. control) in the ischemic cortex 3 and 7 days, respectively, after stroke. vWF expression was also enhanced by 4- and 2.8-fold (vs. control) in the ischemic striatum of G-CSF-treated animals 3 and 7 days, respectively, after stroke; ** p < 0.01, *** p < 0.001, n = 6 rats per group. The data are expressed as mean ± S.E.M. Horizontal bar: 100 μM.

Figure 4

Mobilization of CD34+ cells in the peripheral blood by G-CSF. Top panel: Representative flow cytometry measurements of the number of CD34+ cells in the peripheral blood of vehicle-, tPA-, and G-CSF+tPA-treated stroked rats obtained 3 days after stroke. Isotype-matched antibodies were used as a control. Bottom: Quantitative analysis of the number of circulating CD34+ cells 3 days after stroke. G-CSF treatment significantly increased the number of CD34+ cells in the peripheral blood. The number of CD34+ cells in animals which did not undergo stroke surgery (sham) is also shown; * p < 0.05, n = 7–8 rats per group. The data are expressed as mean ± S.E.M.