RESUMEN
We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions. We found the characteristic protective effects of cyclosporine A (10(-6) M), a known blocker for mitochondrial permeability transition, and SEA0400 (10(-6) M), a blocker for Na(+)/Ca(2+) exchanger, on the abnormal Ca(2+) increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Isquemia/patología , Mitocondrias/metabolismo , Neuronas/ultraestructura , Compuestos de Anilina/farmacología , Animales , Benzofuranos , Citosol/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células HeLa , Compuestos Heterocíclicos con 3 Anillos , Humanos , Imidazoles , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Éteres Fenílicos/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Factores de TiempoRESUMEN
Using laser speckle imaging (LSI), which can visualize quadratic distribution of blood flow, we measured blood flow changes in transient cerebral ischemic mice, and compared these results with data obtained using laser Doppler flowmetry (LDF). In addition, we examined the relationship between ischemic damage and blood flow change. ICR mice (n = 22) were subjected to transient middle cerebral artery occlusion using a 6-0 monofilament under general anesthesia. LSI was performed before -ischemia, during ischemia, and 30 min, 3 h, 24 h, 7 days, and 28 days after ischemia. LDF was monitored continuously from pre-ischemia to 10 min after ischemia commenced. The level of cerebral blood flow (CBF) measured by LSI was less than that using LDF. LSI was able to measure CBF quantitatively and repeatedly. Blood flow -measurements using LSI revealed that recovery of cerebral cortical blood flow after ischemia in mice without cortical infarction was earlier than that seen in mice with cortical infarction. This study indicates that LSI is a -useful technique for analyzing the relationship between -tissue damage and cerebral blood flow change following cerebral ischemia.
Asunto(s)
Circulación Cerebrovascular/fisiología , Diagnóstico por Imagen , Ataque Isquémico Transitorio/diagnóstico , Ataque Isquémico Transitorio/fisiopatología , Rayos Láser , Análisis de Varianza , Animales , Modelos Animales de Enfermedad , Flujometría por Láser-Doppler , Masculino , Ratones , Ratones Endogámicos ICR , Examen Neurológico , Factores de TiempoRESUMEN
BACKGROUND: Although protein kinase C-γ (PKC-γ) is a target for the effects of volatile anesthetics, the molecular mechanisms of the kinase function remain unclear. We examined the effects of different types of anesthetics on PKC-γ knockout mice, and investigated the dynamics of the kinase in mouse brain. METHODS: We measured the required number of times for loss of righting reflex (rtfLORR) after administration of isoflurane, sevoflurane, and propofol on PKC-γ knockout mice and compared with those of wild-type mice. We also used immunoblotting to investigate the intracellular distribution of PKC-γ and phosphorylated PKC-γ (p-PKC-γ) in brain of wild-type mice anesthetized by these anesthetics. RESULTS: Isoflurane and sevoflurane significantly prolonged the rtfLORRs in PKC-γ knockout mice compared with those in wild-type mice, while no significant difference was observed between knockout and wild-type mice treated with propofol. Examination of the cellular fractions showed that PKC-γ was significantly decreased, whereas p-PKC-γ was significantly increased in the synaptic membrane fraction (P2). There was no significant change in the supernatant fraction (S). In propofol-treated mice, PKC-γ and p-PKC-γ showed no significant changes in P2 or S. CONCLUSION: Our results provide new evidence to support the possibility of the involvement of PKC-γ in the actions of volatile anesthetics.
Asunto(s)
Anestésicos por Inhalación/administración & dosificación , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Reflejo de Enderezamiento/efectos de los fármacos , Animales , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/genética , Proteína Quinasa C/deficiencia , Reflejo de Enderezamiento/genética , Factores de TiempoRESUMEN
Traumatic brain injury and ischemia can result in marked neuronal degeneration and residual impairment of cerebral function. However, no effective pharmacological treatment directed at tissues of the central nervous system (CNS) for acute intervention has been developed. The detailed pathophysiological cascade leading to -neurodegeneration in these conditions has not been elucidated, but cellular calcium overload and mitochondrial dysfunction have been implicated in a wide range of animal models involving degeneration of the CNS. In particular, activation of the calcium-induced mitochondrial permeability transition (mPT) is considered to be a major cause of cell death inferred by the broad and potent neuroprotective effects of -pharmacological inhibitors of mPT, especially modulators of cyclophilin activity and, more specifically, genetic inactivation of the mitochondrial cyclophilin, cyclophilin D. Reviewed are evidence and challenges that could bring on the dawning of mitochondrial medicine aimed at safeguarding energy supply following acute injury to the CNS.
Asunto(s)
Ciclofilinas/metabolismo , Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores , Animales , Arsenicales/farmacología , Arsenicales/uso terapéutico , Lesiones Encefálicas/tratamiento farmacológico , Calcio/metabolismo , Peptidil-Prolil Isomerasa F , Ciclosporina/farmacología , Ciclosporina/uso terapéutico , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Humanos , Isquemia/tratamiento farmacológicoRESUMEN
BACKGROUND: Recently, various sets of protein -biomarkers have been discovered in important diseases such as cancers, brain stroke and heart attack. However, clinical validation is difficult and time-consuming by individual assays or because of very low concentrations at early stages of the diseases. We have developed assay technology through an innovative modification of the immuno-PCR method for the super-sensitive and multiplex detection of target biomarkers. METHODS: In the assay technology, each different oligo-tag simultaneously detects multiplex protein targets with extremely high-level sensitivity in a dose-dependent manner by qRT-PCR (maximum: three plexes). In this study, we measured specific secreted protein concentrations in the culture supernatant of a 24-h culture of transfected SH-SY5Y cells with MUSTag. RESULTS: There was a significant increase in the protein level of tumor necrosis factor (TNF)-α measured with extremely high-level sensitivity (≥10 pg/mL). Compared with negative controls, the levels of TNF-α increased from 16.9 to 28.1 pg/mL (p = 0.011). CONCLUSION: We suggest that our assay technology might be of clinical value in treating patients with cancer, cerebral ischemia, or patients who need a prompt and predictive diagnosis for adequate treatment.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Factor de Necrosis Tumoral alfa/análisis , Angiopoyetina 1/genética , Angiopoyetina 1/metabolismo , Línea Celular Tumoral , Factor 3 de Iniciación Eucariótica/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Neuroblastoma , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transfección , Factor de Crecimiento Transformador betaRESUMEN
Anesthetic agents, especially, volatile anesthetics are considered to exert organ toxicity such as nephrotoxicity and hepatotoxicity; however, recent aggressive researches explored the beneficial effects of volatile anesthetics as an organ protectant. Ischemic preconditioning is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the myocardium and brain against prolonged ischemic insult. General anesthesia showed the protection against both ischemic myocardial and brain reperfusion injuries. This phenomenon is called anesthetic preconditioning. Regarding the organ protection, anesthetic preconditioning is one of the useful ways to diverse the organ protective effects not only to heart but also brain. Nowadays, ischemic postconditioning, consisting of repeated brief cycles of ischemia-reperfusion performed immediately after reperfusion following a prolonged ischemic insult, dramatically reduces infarct size in experimental models and such clinical studies are reported. Both preconditioning and postconditioning share the same signal transduction pathway and inhibit the mitochondrial permeability transition (MPT) that leads to either apoptosis or necrosis of myocardium and neuronal cell. Both phenomena look very promising, but we still lack the real evidence for human reserach in terms of the clinical outcome and further analysis is necessary. Neurotoxicities of anesthetic agents are very crucial problems for the patient and they are considered to be due to the activation of IP3 receptor in ER after exposure to volatile anesthetics. Massive release of Ca2+ from ER induces Ca2+ overload leading to mitochondria permeability transition (MPT) and induces apoptosis in the brain or aggravates the neurodegenerative disease. Susceptible mechanisms and beneficial treatment for the toxicity of general anesthesia is considered as a critical subject to discuss and challenge to solve for our future.