RESUMEN
Cation channels conduct calcium, sodium and potassium, cations that are likely deleterious in the evolution of focal ischemic injury. We studied the effects of a novel, broad-spectrum inhibitor of several cation channels, LOE 908 MS, on acute ischemic lesion development with diffusion-weighted magnetic resonance imaging (DWI) and on cerebral perfusion with perfusion imaging (PI) in vivo and on cerebral infarct size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem. A total of 18 male Sprague-Dawley rats underwent 90 min of middle cerebral artery occlusion (MCAO) and were randomly and blindly assigned to either LOE 908 MS or vehicle starting 30 min after inducing focal ischemia and continuing for 4 h. Whole-brain DWI and multislice PI were done before initiation of treatment and repeated frequently for the next 3.5 h. DWI-derived lesion volume at 4 h showed a significant difference in favor of the drug treated group (P=0.03), whereas PI-derived perfusion deficit volumes did not significantly differ between the groups. The postmortem infarct volume at 24 h was significantly attenuated in the treated group in comparison to controls (P=0.0001) and neurological score was significantly better in the treated group (P<0.02). Blocking several distinct cation channels with LOE 908 MS significantly reduced infarct size and improved neurological outcome without observable adverse effects in this focal ischemia model.
Asunto(s)
Acetamidas/farmacología , Isquemia Encefálica/patología , Infarto Cerebral/patología , Canales Iónicos/efectos de los fármacos , Isoquinolinas/farmacología , Acetamidas/uso terapéutico , Animales , Isquemia Encefálica/complicaciones , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Infarto Cerebral/prevención & control , Isoquinolinas/uso terapéutico , Imagen por Resonancia Magnética , Masculino , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Cambios Post Mortem , Ratas , Ratas Sprague-Dawley , Veratridina/farmacologíaRESUMEN
We have synthesized a new benzomorphan derivative, 2R-[2alpha,3(S*), 6alpha]-1,2,3,4,5,6-hexahydro-6,11, 11-trimethyl-3-[2-(phenylmethoxy)propyl]-2, 6-methano-3-benzazocin-10-ol hydrochloride (BIII 890 CL), which displaced [(3)H]batrachotoxinin A-20alpha-benzoate from neurotoxin receptor site 2 of the Na(+) channel in rat brain synaptosomes (IC(50) = 49 nM), but exhibited only low affinity for 65 other receptors and ion channels. BIII 890 CL inhibited Na(+) channels in cells transfected with type IIA Na(+) channel alpha subunits and shifted steady-state inactivation curves to more negative potentials. The IC(50) value for the inactivated Na(+) channel was much lower (77 nM) than for Na(+) channels in the resting state (18 microM). Point mutations F1764A and Y1771A in transmembrane segment S6 in domain IV of the alpha subunit reduced the voltage- and frequency-dependent block, findings which suggest that BIII 890 CL binds to the local anesthetic receptor site in the pore. BIII 890 CL inhibited veratridine-induced glutamate release in brain slices, as well as glutamate release and neurotoxicity in cultured cortical neurons. BIII 890 CL (3-30 mg/kg s.c.) reduced lesion size in mice and rats when administered 5 min after permanent focal cerebral ischemia at doses that did not impair motor coordination. In contrast to many other agents, BIII 890 CL was neuroprotective in both cortical and subcortical regions of the rat brain. Our results demonstrate that BIII 890 CL is a potent, selective, and highly use-dependent Na(+) channel blocker that protects brain tissue from the deleterious effects of focal cerebral ischemia in rodents.
Asunto(s)
Benzomorfanos/farmacología , Encéfalo/fisiología , Ataque Isquémico Transitorio/fisiopatología , Fármacos Neuroprotectores/farmacología , Bloqueadores de los Canales de Sodio , Sinaptosomas/fisiología , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , Unión Competitiva , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiología , Ácido Glutámico/metabolismo , Ataque Isquémico Transitorio/prevención & control , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos , Actividad Motora/efectos de los fármacos , Ratas , Ratas Wistar , Proteínas Recombinantes/metabolismo , Canales de Sodio/química , Canales de Sodio/fisiología , Transfección , Veratridina/farmacologíaRESUMEN
Cation channels conduct calcium, sodium, and potassium, cations that are likely deleterious in the evolution of focal ischemic injury. Diffusion-weighted magnetic resonance imaging (DWI) is a powerful tool for evaluation of acute cerebral ischemia. We studied the effects of a novel, broad-spectrum inhibitor of several cation channels, LOE 908 MS, on acute ischemic lesion development with DWI and on cerebral infarct size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem. Eighteen male Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO) and were randomly and blindly assigned to either LOE 908 MS (1 mg/kg bolus 30 min after MCAO and continuous i.v. infusion of 10 mg/kg for 4 h thereafter) or vehicle. Whole-brain DWI was done before initiation of treatment and repeated every 30 min for the next 3.5 h. The animals were reperfused in the magnetic resonance imaging (MRI) scanner 90 min after MCAO. At 24 h, the animals were killed, and the brains were cut into six 2-mm-thick slices and stained with 2% TTC. Percent hemispheric lesion volume (%HLV) was calculated for each animal. Physiological parameters, body weight, and premature mortality (3 in the placebo group and 1 in the treated group) did not differ between the groups. No hypotension, abnormal behavior, or other adverse effects were seen. Pretreatment, the DWI-derived %HLV did not differ between the groups (19.8 +/- 6.2 in the control group and 17.9 +/- 7.9 in the treated group), whereas at 4 h after MCAO, it was significantly smaller in the treated group (21.8 +/- 15.4 vs 40.4 +/- 15.5, p = 0.03). Postmortem, TTC-derived %HLV was significantly attenuated in the LOE 908 MS group (21.3 +/- 11.9 vs 50.1 +/- 10.7, p = 0.0001) and the neurological scores at 24 h were significantly better among the treated rats (2.1 +/- 1.5 vs 4.0 +/- 1.0, p < 0.02). LOE 908 MS significantly improved neurological outcome and reduced infarct size without observable effects in rats as demonstrated in vivo by DWI and confirmed postmortem by TTC staining. Blocking several distinct cation channels by LOE 908 MS showed significant neuroprotection.
Asunto(s)
Acetamidas/farmacología , Edema Encefálico/fisiopatología , Infarto de la Arteria Cerebral Media/fisiopatología , Isoquinolinas/farmacología , Bloqueadores de los Canales de Potasio , Bloqueadores de los Canales de Sodio , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Difusión , Aumento de la Imagen , Imagen por Resonancia Magnética , Masculino , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Canales de Sodio/fisiología , Equilibrio Hidroelectrolítico/efectos de los fármacos , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
OBJECTIVE: We investigated the influence of dose, route and time of administration on the neuroprotective effects of the noncompetitive N-methyl-D-aspartic acid antagonist BIII 277 CL ([2R-[2alpha, 3(R*), 6alpha]]-1,2,3,4,5,6-hexahydro-3-(2-methoxy-propyl)-6,11,11-trimethyl-2,6-methao-3-benzazocin-9-ol hydrochloride). METHODS: Focal cerebral ischemia was induced in isoflurane-anaesthetized Fischer rats by permanent occlusion of the left middle cerebral artery. Rats were treated with BIII 277 CL three times at doses of 1 and 3 mg/kg intraperitoneally (IP) (5 to 10 minutes and 4 and 24 hours after occlusion) or twice with 0.1, 0.3, and 1.0 mg/kg subcutaneously (SC) (5 to 10 minutes and 3 hours after occlusion) or twice with 1 mg/kg SC (30 minutes and 3 hours 30 minutes; 1 and 4 hours; 2 and 5 hours; or 4 and 7 hours after occlusion). Other rats received (+)MK-801 (dizocilpine) three times at doses of 0.3, 1.0, and 3.0 mg/kg IP (5 to 10 minutes and 4 and 24 hours after occlusion). Control rats received an equal volume of saline. Infarct volume was determined 48 hours after occlusion by standard histological techniques. RESULTS: IP administration of BIII 277 CL caused a dose-dependent reduction of infarct volume (1 mg/kg, 13%; 3 mg/kg, 25%). (+)MK-801 had similar effects (0.3 mg/kg, 13%; 1.0 mg/kg, 21%; 3 mg/kg, 27%). BIII 277 CL also dose-dependently reduced the infarct volume after SC administration (0.1 mg/kg, 14%; 0.3 mg/kg, 30%; 1.0 mg/kg, 28%). Furthermore, significant neuroprotective effects of BIII 277 CL were observed even when initial treatment was delayed up to 1 hour after occlusion (30 minutes, 28%; 1 hour, 23%; 2 hours, 5%; 4 hours, 4%). CONCLUSIONS: These results indicate that BIII 277 CL shows significant neuroprotective effects at doses as low as 0.1 mg/kg SC. The effects after IP administration are comparable with those of (+)MK-801, and significant effects were observed even when the BIII 277 CL was first administered up to 1 hour after the beginning of ischemia.
RESUMEN
We have investigated the relationship between energy metabolism, NMDA-receptor antagonism, and anoxic damage in vitro. Anoxic damage was assessed by measuring protein synthesis, defined as the incorporation of [14C]lysine into perchloric acid-insoluble tissue extracts. The concentrations of energy metabolites were measured by ion-exchange HPLC. Anoxia caused an inhibition of protein synthesis, a reduction in phosphocreatine and adenosine triphosphate, and extensive neuronal damage. The reduction of protein synthesis depended on the duration of anoxia and the time allowed for recovery. Preincubation with the creatine dose-dependently (0.03-3 mmol/L) increased baseline levels of phosphocreatine, reduced the anoxia-induced decline in phosphocreatine and adenosine triphosphate, prevented the impairment of protein synthesis, and reduced neuronal death. Incubation with (R,S)-3-guanidinobutyric acid, a synthetic analogue of creatine that cannot be phosphorylated, did not prevent the anoxia-induced impairment of protein synthesis and did not enhance the levels of phosphocreatine and adenosine triphosphate. Incubation with a combination of both creatine and the noncompetitive NMDA antagonist MK-801 provided complete protection. These results indicate that energy status is a major factor controlling anoxic damage in the rat hippocampal slice.
Asunto(s)
Creatina/farmacología , Hipocampo/metabolismo , Hipocampo/patología , Hipoxia/patología , Fármacos Neuroprotectores/farmacología , Fosfocreatina/metabolismo , Animales , Creatina/antagonistas & inhibidores , Metabolismo Energético/efectos de los fármacos , Guanidinas/farmacología , Hipocampo/efectos de los fármacos , Hipoxia/metabolismo , Técnicas In Vitro , Masculino , Biosíntesis de Proteínas , Ratas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidoresRESUMEN
Magnetic resonance imaging has been used to follow the time course of lesions induced in the rat brain as an animal model for characterization of the volume of the lesion. The dispersion in spin-spin relaxation has been used to characterize the nature of the brain lesion. Parameter selective estimation of T2, quantitative determination of the lesion size and volume selective in vivo proton spectroscopy have been employed for the purpose. The work has been carried out on rats which were subject to lesioning by ibotenic acid as a model for excitotoxicity and also on rats which received doses of ibotenic acid and subsequent doses of the NMDA antagonist drug MK 801 (dizocilpine). The time course of the progress of the lesions in untreated animals and the effect of neuroprotection by MK 801 was continuously monitored in all test animals. Further, a relatively new inhalation anesthetic agent, isoflurane, has been employed. A more logical and semiquantitative T2 bandwidth demarkation useful in distinguishing different degrees of lesioning from the onset and up to the 'edema' stage through penumbra (mild lesion), medium degree lesion and severe lesion has been proposed.