Effect of Long-Term Treatment with Fimasartan on Transient Focal Ischemia in Rat Brain

Abstract

Fimasartan is a newly developed angiotensin receptor blocker, which may have protective effects during myocardial infarction or atherosclerosis. In this context, we investigated the effects of long-term treatment with low-dose fimasartan on focal ischemia in rat brain. We induced focal ischemia in brain by transient intraluminal occlusion of middle cerebral artery (MCA) and administered low-dose (0.5 mg/kg) or regular doses (1 or 3 mg/kg) of fimasartan via intravenous routes. After the administration of low-dose (0.5 mg/kg) fimasartan, blood pressure did not decrease compared to the phosphate-buffered saline- (PBS-) control with MCA occlusion (MCAO) group. The infarct volume and ischemic cell death were reduced in the low-dose fimasartan-treated group (46 ± 41 mm(3) for 0.5 mg/kg and 153 ± 47 mm(3) for PBS-control with MCAO; P < 0.01) but not in the regular-dose groups. Low-dose fimasartan treatment improved functional recovery after ischemia and significantly decreased mortality. In our study, fimasartan reduced the degradation of IκB and the formation of an inflammatory end-product, COX-2. As a result, the recruitment of inflammatory cells in the peri-infarct area decreased in fimasartan-treated group. We have demonstrated that long-term, low-dose fimasartan treatment improved outcomes after focal ischemia in the brain via a reduction of inflammation.

Figure 1

Schematic diagram of the study protocols. For Sprague-Dawley rats, low-dose (0.5 mg/kg) or regular doses (1 or 3 mg/kg) were administered for 4 weeks via an oral route before the induction of transient middle cerebral artery (MCA) occlusion. At 2 days, western blot (WB) analyses were performed. At 7 days immunofluorescent staining was performed, and infarct volume was measured. Behavior tests were performed for 14 days after induction of transient MCA occlusion, and mortality was censored at 28 days. Blood pressure was monitored during the pretreatment and follow-up period.

Figure 2

Blood pressure. (a) Mean blood pressures (MBPs) were decreased in the regular-dose fimasartan groups at 3 days after fimasartan administration. The MBPs in the low-dose fimasartan group were not different from the PBS-control with MCAO group (n = 9 each). (b) Invasive systolic blood pressures decreased in the regular-dose groups but did not decrease in the low-dose fimasartan group (n = 3 each). (c) Invasive MPBs in the low-dose fimasartan did not decrease compared to PBS-controls with MCAO (n = 3 each). (d) Invasive diastolic blood pressures decreased in the regular-dose groups but were not reduced in the low-dose group than PBS-control with MCAO group (n = 3 each).

Figure 3

Measurements of infarct volume and ischemic cell death. (a) Sham operations did not make any infarct lesions in the brain (n = 6). The infarct volume in the low-dose fimasartan group (0.5 mg/kg, n = 8) decreased significantly compared to the PBS-control with MCAO group (P < 0.05 by Bonferroni correction, n = 8), but there was no difference between the regular-dose group and PBS-controls with MCAO (P for 1 mg/kg = 0.21 and P for 3 mg/kg = 0.12; n = 6 each). (b) Ischemic cell death, evaluated by TUNEL staining, decreased in the low-dose fimasartan group compared with the PBS-control with MCAO group (P < 0.05; n = 4 each). Representative TUNEL-positive cells (dark brown color) are indicated by black arrows, and TUNEL-negative cells are counterstained by methyl green (blue color). Scale bar = 50 μm.

Figure 4

Functional recovery and mortality after focal cerebral ischemia. (a) Low-dose fimasartan improved functional recovery after transient focal ischemia compared to PBS-controls with MCAO. This improvement was prominent at 14 days after ischemia (P < 0.05; n = 17 for 0.5 mg/kg and n = 12 for control). (b) The mortality rate decreased significantly in the low-dose fimasartan group compared with PBS-controls with MCAO (P by log-rank test < 0.05; n = 18 each).

Figure 5

Immunofluorescent staining of inflammatory cells and western blot analyses. (a) Ox6-stained microglia/macrophages (green) were less frequently found in the periphery of the lesion, and the density in the fimasartan-treated group was lower than the PBS-control with MCAO group (n = 4 each). Scale bar = 50 μm. (b) According to the quantitative analysis, the fimasartan-injected group exhibited a lower number of Ox6+ cells (P < 0.05) compared to the PBS-control with MCAO group. (c) At 48 hours after ischemia induction, the density of IκB increased and cyclooxygenase-2 (COX-2) decreased in the fimasartan-treated group compared with the PBS-control with MCAO group (n = 4 each). (d) In the quantitative analyses, the degradation of IκB was suppressed in the low-dose fimasartan-treated group in the lesion and was comparable to the contralateral brain in the PBS-control with MCAO group compared with lesion side in the PBS-control with MCAO group (P < 0.05). (e) The density of COX-2 in the infarcted hemisphere was 1.6-fold higher in the PBS-control with MCAO group compared to the fimasartan-treated group (P < 0.05).