RESUMEN
A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.
Asunto(s)
Química Encefálica , Cromatografía Líquida de Alta Presión/métodos , Esfingomielinas/análisis , Animales , Dimiristoilfosfatidilcolina/normas , Ratas , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
PURPOSE: To determine if posthypoxia treatment with erythropoietin (EPO) has protective effects against subsequent susceptibility to seizure related neuronal injury in rat pups subjected to acute hypoxia at P10. METHODS: Four groups of rats were manipulated at P10, as described below, then all received kainic acid (KA) (10 mg/kg i.p.) at P29: Hypoxia-NS-KA group (n = 11): subjected to acute hypoxia (down to 4% O2), and then immediately received saline i.p. Hypoxia-EPO-KA group (n = 10): subjected to acute hypoxia and then immediately received EPO (1,000 U/Kg i.p.). Normoxia-NS-KA group (n = 11): sham manipulated and injected with saline. Normoxia-EPO-KA group (n = 10): sham manipulated then immediately injected with EPO (1000 U/Kg i.p.). After receiving KA at P29, all rats were monitored using videotape techniques, and were sacrificed at P31. TUNEL and Hoechst stains to assess for apoptosis, and regular histology for hippocampal cell counts were performed. RESULTS: Administration of the single dose of erythropoietin directly after an acute hypoxic event at P10 resulted at P29 in increased latency to forelimb clonus seizures, reduced duration of these seizures, protection against hippocampal cell loss, and decreased hippocampal apoptosis in the Hypoxia-EPO-KA group as compared to the Hypoxia-NS-KA group. CONCLUSION: These data support the presence of favorable protective effects of erythropoietin against the long-term consequences of acute hypoxia in the developing brain and raise the possibility of its investigation as a potential neuroprotective agent after human neonatal hypoxic encephalopathy.
Asunto(s)
Eritropoyetina/farmacología , Hipoxia/metabolismo , Convulsiones/inducido químicamente , Convulsiones/prevención & control , Animales , Animales Recién Nacidos , Recuento de Células , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/metabolismo , Relación Dosis-Respuesta a Droga , Eritropoyetina/administración & dosificación , Agonistas de Aminoácidos Excitadores/farmacología , Hipocampo/patología , Humanos , Hipoxia/patología , Hipoxia Encefálica/prevención & control , Ácido Kaínico/farmacología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas Sprague-Dawley , Convulsiones/patologíaRESUMEN
Ten-day-old rat pups (P10) subjected to acute hypoxia (down to 4% O2) had as adults increased aggression (handling test), memory impairment (water maze test), and decreased CA1 cell counts. Pups subjected to chronic hypoxia (10% O2 from P0 to P21) had increased aggression, hyperactivity (open-field test), and decreased CA1 cell counts. Chronic hypoxia with superimposed acute hypoxia resulted in consequences that were not different from those of chronic hypoxia.