A rho-kinase inhibitor prevents apoptosis-induced neuronal cell death following transient focal ischemia in rats

Background and aims: Recently, it has been reported that rho-kinase that participates in the regulation of vascular tonus, spasm, and remodeling in response to smooth muscle constriction, is involved in the atherosclerosis, inflammation, and hypertention (1). Furthermore, rho-kinase is involved in nitric oxide (NO), free radical, resulting in the neuronal cell death (2). Nevertheless, the mechanism of rho-kinase on the ischemic brain have not been fully understood. The aim of this study is, therefore, to determine whether a newly rho-kinase inhibitor, fasudil, which is a myosin light chain kinase inhibitor and have strong inhibition of rho-kinase, would prevent neuronal cell death following transient focal ischemia in rats compared to a free radical scavenger, edaravone , which is the first clinical neuroprotective agent used in Japan for the treatment of acute stroke patients. Methods: Sprague-Dawley rats were subjected to MCAo using an intraluminal suture technique for 90 minutes (3). The rats were reperfused for 24 hours and decapitated for infarct and edema analysis (4). Animals were randomly divided into the following four groups. (I) vehicle-treated control group (II) low dose of fasudil (3.0 mg/kg)-treated group (III) high dose of fasudil (10.0 mg/kg)-treated group (IV) edaravone-treated group. A rho-kinase inhibitor-treated animals received a continuous injection of fasudil (3.0 or 10.0 mg/kg) intravenously for 60 minutes after the onset of ischemia, while vehicle-treated groups received same dose of vehicle. Edaravone-treated animals received twice injection of edaravone (3.0 mg/kg) intravenously immediately after the reperfusion and 30 minutes after reperfusion. During ischemia, temporal muscle and rectal temperatures were monitored and maintained at 37 degrees in the experimental animals. Neurological symptom evaluations (posture and hemiplegia) were performed immediately before infarct and edema analysis. Furthermore, cortical cerebral blood flow was measured during ischemia and reperfusion. Results: The cortical and striatal infarct volume in the edaravone-treated group was significantly less than those in the vehicle-treated control group I (p<0.001, p<0.01 respectively). Fasudil (group II, III) decreased the cortical or striatal infarct volume significantly compared with those of groups I (p<0.001, p<0.05 respectively). The cortical or striatal edema significantly was also improved in the low dose (3.0 mg/kg) fasudil-treated groups significantly, compared with those of groups I (p<0.05, p<0.05, respectively). Moreover, low dose or high dose (10.0 mg/kg) fasudil significantly improved neurological symptoms (p<0.001, p<0.01 respectively). Conclusions: These results demonstrate that a rho-kinase inhibitor, fasudil prevents neuronal cell death, compared to a free radical scavenger, edaravone. Furthermore, it is suggested that this rho-kinase inhibitor may be a candidate for new neuroprotectant for the treatment of acute stroke in future.