Neuroprotective potential of fasudil mesylate in brain ischemia-reperfusion injury of rats

Abstract

We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757-768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion.

Fig. 1

Effects of fasudil mesylate (FM) on cerebral infarct size and neurological score after 2 h MCAO and 24 h reperfusion (n = 10). (a) Representative coronal brain sections stained with 2% TTC from sham, vehicle and FM-treated groups, respectively. (b) Quantitative analysis of cerebral infarct size from vehicle and FM-treated groups (^## P < 0.01 vs. vehicle group). (c) Neurological deficit score was measured at 6 and 26 h after MCAO (^## P < 0.01 vs. vehicle group). Sham group includes rats without MCAO, vehicle group is NS-treated rats with MCAO and FM-treated group FM-treated (10 mg/kg i.p.) rats with MCAO

Fig. 2

Effect of fasudil mesylate (FM) on morphologic changes in ischemic regions of rat cortex induced by 2 h MCAO and 24 h reperfusion. The different treatments of animals are shown in (a) the cortex tissues from the rats without MCAO (sham group); (b) the tissues from NS-treated rats with MCAO (vehicle group); (c) the tissues from FM-treated rats with MCAO (FM-treated group). Fasudil mesylate treatment (10 mg/kg i.p.) markedly attenuated these pathological and morphological changes as compared with vehicle group

Fig. 3

Immunohistochemistry graphs show the expression of eNOS (upper panel) and iNOS (lower panel) in ischemic regions of rat cortex after 2 h MCAO and 24 h reperfusion. In the figures for both eNOS and iNOS immunoreactivities, (a) is sham group for rats without MCAO; (b) is vehicle group treated with NS in rats with MCAO; and (c) is FM-treated rats with MCAO (FM10 mg/kg i.p.). Arrows represent eNOS and iNOS positive cells, respectively (×400)

Fig. 4

Western blot analysis demonstrates the expression of eNOS (upper panel in a) and iNOS (lower panel in a) in ischemic regions of rat cortex after 2 h MCAO and 24 h reperfusion. (a) Western blot analysis showing protein bands of rat cortex from each group; the corresponding β-actin bands as controls shown in the same blot (upper lane). (b) After densitometric quantification, the data are expressed as mean ± SEM (* P < 0.05 vs. sham; ^#  P < 0.05 vs. vehicle). Each group designated as sham, vehicle and FM-treated, was described as in Fig. 3

Fig. 5

Effects of fasudil mesylate (FM) on the viability of rat brain cortical and hippocampal slices after OGD insult in vitro. OD₄₉₀ value of TTC staining (A) was decreased but LDH efflux (B) increased in OGD group, whereas fasudil mesylate (10, 100 μmol/l) significantly attenuated these changes (P < 0.05). The data are expressed as mean ± SEM (n = 8). Compared with the control group, fluorescent intensity of PI staining (C) was significantly increased in OGD group, whereas fasudil mesylate at both 10 and 100 μmol/l decreased PI fluorescent intensity of cortical and hippocampal slices after OGD insult (P < 0.05). The data are expressed as mean ± SEM. (n = 5). For (A–C), *** P < 0.001 vs. control group; ^#  P < 0.05, ^##  P < 0.01, and ^###  P < 0.001 vs. OGD group. In the bottom are representative images of PI staining (D) for cortical and hippocampal slices (×10). These graphs are (a and e) control slices without OGD; (b and f) OGD slices; (c and g) OGD slices + FM 10 μM; (d and h) OGD slices + FM 100 μM

Fig. 6

Effects of fasudil mesylate (FM) on iNOS and SOD activity of rat cortical and hippocampal slices after OGD insult (n = 8). The data are expressed as mean ± SEM (P < 0.05). *** P < 0.001 vs. control group; ^#  P < 0.05, ^##  P < 0.01, and ^###  P < 0.001 vs. OGD group. Each group was treated as described in the method section