Supra-Additive Neuroprotection by Renexin, a Mixed Compound of Ginkgo Biloba Extract and Cilostazol, Against Apoptotic White Matter Changes in Rat after Chronic Cerebral Hypoperfusion

BACKGROUND AND PURPOSE: White-matter (WM) lesions are known to potentiate cognitive impairment in poststroke patients. The present study was designed to assess whether Ginkgo biloba extract (GB) and cilostazol, which were evaluated alone and in a combination formula (Renexin), can attenuate the WM lesions and cognitive decline caused by chronic hypoperfusion in the rat. METHODS: Animals were divided into five treatment groups: cilostazol (25 mg/kg/day), GB (20 mg/kg/day), Renexin (25 mg/kg/day cilostazol + 20 mg/kg/day GB), vehicle, and sham. The animals received the treatments orally 1 day after bilateral common carotid artery occlusion [two-vessel occlusion (2VO); except for the sham group, which underwent the surgery but the arteries were not occluded], and then the same dose every day for 21 days thereafter. Prior to sacrificing the rats, repetitive eight-arm radial maze testing was performed to examine their cognitive abilities. After drug administration and cognitive testing, brain tissues were isolated for Klüver-Barrera and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL) staining, immunohistochemical assessment of glial fibrillary acidic protein (GFAP) and CD11b (OX-42), and to assay free-radical scavenging activity. RESULTS: We found that the significant WM lesions induced by 2VO was ameliorated significantly by treatment with cilostazol, GB, and Renexin, in association with increased TUNEL-positive cells. In addition, chronic cerebral hypoperfusion caused a large increase in the degree of GFAP and OX-42 immunoreactivity and free-radical activity in the optic tract. These abnormalities were significantly reversed by the three drugs, but most prominently by Renexin, suggesting a markedly enhanced or supra-additive effect of cilostazol and GB when administered together. CONCLUSIONS: Significant attenuation of cytoarchitectural damage and apoptotic cell death was found with GB and cilostazol, but a markedly enhanced effect was seen for treatment with their combination in the WM of rat brains after bilateral occlusion of the common carotid arteries. We suggest that combination therapy with GB and cilostazol provides enhanced neuroprotective effects and induces subsequent cognitive improvement in patients with chronic ischemic conditions.