RESUMEN
Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.
Asunto(s)
Encéfalo/enzimología , Infarto Cerebral/enzimología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Factor de Transcripción Activador 3/metabolismo , Amidas/farmacología , Animales , Western Blotting , Encéfalo/patología , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Infarto Cerebral/patología , Inhibidores Enzimáticos/farmacología , Hipocampo/enzimología , Hipocampo/patología , Inmunohistoquímica , Masculino , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Extractos de Tejidos/química , Extractos de Tejidos/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.
Asunto(s)
Isquemia Encefálica/enzimología , Isquemia Encefálica/etiología , Trombosis Intracraneal/complicaciones , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/administración & dosificación , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/metabolismo , Animales , Antipirina/análogos & derivados , Velocidad del Flujo Sanguíneo/efectos de los fármacos , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/patología , Infarto Cerebral/tratamiento farmacológico , Infarto Cerebral/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Trombosis Intracraneal/inducido químicamente , Trombosis Intracraneal/metabolismo , Trombosis Intracraneal/terapia , Ácidos Láuricos , Masculino , Miosinas/metabolismo , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Factores de TiempoRESUMEN
The neuroprotective potential and therapeutic time window for fasudil, a Rho-kinase inhibitor (RKI), were evaluated for delayed neuronal death in gerbils. A preliminary screening was done on fasudil, ozagrel, and edaravone using a single administration in a delayed neuronal death study. Intraperitoneal (i.p.) administration of edaravone, a free radical scavenger (3, 10 mg/kg) immediately after re-circulation did not reduce neuronal degeneration. We previously reported that ozagrel, a thromboxane A(2) synthetase inhibitor (30 mg/kg) also did not reduce neuronal degeneration, while fasudil (3, 30 mg/kg) significantly protected against the ischemia-induced neuronal loss. To clarify the therapeutic time window of fasudil, which showed a positive effect in a preliminary screening, animals received their first i.p. administration of fasudil (10 mg/kg) 24 or 48 h after ischemia. Administration of fasudil twice daily was continued until day 6. Fasudil significantly protected against the ischemia-induced delayed neuronal death when the treatment was started 24 h after ischemia. In gerbils, hydroxyfasudil, an active metabolite of fasudil, was found following an i.p. administration of fasudil (10 mg/kg), and the value of the area under the plasma level curve of hydroxyfasudil was 7 times higher than that of fasudil. Hydroxyfasudil may contribute to the potency of fasudil. The present findings indicate that the RKI fasudil reduces ischemic neuronal damage with a wide therapeutic time window in gerbil, and may be useful in the treatment of acute ischemic stroke in humans.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Isquemia , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/sangre , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacocinética , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Antipirina/análogos & derivados , Antipirina/uso terapéutico , Muerte Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Edaravona , Depuradores de Radicales Libres/uso terapéutico , Gerbillinae , Isquemia/sangre , Isquemia/complicaciones , Isquemia/tratamiento farmacológico , Isquemia/patología , Masculino , Neuronas/patología , Fármacos Neuroprotectores/sangre , Fármacos Neuroprotectores/farmacocinética , Factores de TiempoRESUMEN
The aim of this study was to develop a new model of vasopressin-induced chronic myocardial damage based on sustained ST-segment depression in electrocardiogram (ECG) with progression of myocardial fibrosis in rats. Furthermore, using this model, we examined the prophylactic potential of fasudil, a Rho-kinase inhibitor, against myocardial damage induced by vasopressin. In 10-week old male Donryu rats, intravenous administration of arginine vasopressin (0.5 iu/kg) induced significant ST-segment depression. Two days and one week after the administration of vasopressin, ST-segment depression was -0.19 +/- 0.02 and -0.14 +/- 0.02 mV, respectively. Fasudil (10 and 30 mg/kg, p.o.) significantly attenuated the ST-segment depression induced by vasopressin. One week after the administration of vasopressin, the percent area of myocardial fibrosis in control animals (0.42 +/- 0.11%, p < 0.01) was significantly greater than that in normal animals (0.05 +/- 0.01%). Fasudil (10 and 30 mg/kg) significantly prevented the development of the fibrosis. We present a new model of chronic myocardial damage based on sustained ST-segment depression with progression of myocardial fibrosis in rats, and suggest that this model may be useful to investigate the treatment of chronic angina. Inhibition of Rho-kinase is efficacious in preventing the ECG change and development of fibrosis induced by vasopressin in this model.