| Hypoxic-ischemic brain damage (HIBD) can result from the perinatal asphyxia period that results clinical encephalopathy with irreversible brain damage and a series of severe nervous sequela in children. Though there exists many theories explaining the pathogenesis mechanism of HIBD, it has not yet been fully elucidated. Objective:To explore the possible protective effect mechanisms of Phenobarbital, the resent study was designed to observe its effect on the neonatal rats with hypoxic-ischemic brain damage and KCC2level in the brain.Methods:Seven-day-old Sprague-Dawley (SD) rat pups were randomly divided into:(1) sham operation group (2) HIBD group (3) Phenobarbital treatment group. Rat HIBD model was prepared according the Rice-Vannucci method by legating the left common carotid artery, followed by exposure to8%oxygen and92%nitrogen for2hours in the HIBD group and the Phenobarbital treatment group. The following treatment protocol:the intraperitoneal bolus dose of25mg/kg Phenobarbital, which was used after HIBD, was followed18h later by an administration of15mg/kg (i.p.), and then twice daily (between7:00and8:00A.M. and5:00and6:00P.M.)15mg/kg (i.p.) for the subsequent3days. Sham operation group received injection with saline during the corresponding periods.2,3,5-triphenyltetrazolium chloride (TTC) was preformed to evaluate the infarct ratio of hypoxic-ischemic; Spontaneous activity test and Morris water maze test were conducted for the long-term neurological and neurobehavioral functions; Real-time PCR was used to measure KCC2mRNA levels; Western blot analysis and Immunohistochemistry was employed to determine alterations of KCC2in the hypoxic-ischemic brain.Results:(1) Two hours after hypoxic-ischemic, neonatal rats appear fixed to the left rotates, the symbol behavior of successful models.(2) TTC shows that, HIBD group has clearly cerebral infarction area. This also proves that we have made successful models. Comparison with the HIBD group, Phenobarbital intraperitoneally can reduce the cerebral infarction ratio (p<0.01) (3) The Spontaneous activity test indicated that all rats experienced hypoxic-ischemic insult moved less with comparison of sham operation group (p<0.05).However, the rats in Phenobarbital treatment group were more active and not different from sham operation group (p>0.05).(4) Morris water-maze showed that, HIBD group>Phenobarbital treatment group>sham operation group for the average escape latency on different time were with significance; Compared with Phenobarbital treatment group and sham operation group, the percentage time of Target quadrant and number of crossing the platform in HIBD rats were significant.The result suggested that HIBD rats had learning and memory function damage, and early Phenobarbital treatment could improve long-term neurobehavioral.(5) Compared with Sham group, Expression of KCC2mRNA and protein is acute substantially down-regulated in HIBD group, the down-regulation of KCC2significantly appears in hippocampus CA1. Compared with HIBD group, that of Phenobarbital treatment group is with similar distribution but less down-regulation. There are significant statistic differences between these two groups (p<0.01)Conclusion:(1) We observed that Phenobarbital treatment reduced the infarct ratio, improved the spontaneous activity and the hippocampal memory function. It suggested that Phenobarbital treatment significantly palliated the hypoxic-ischemic pathogenesis process, attenuated neonatal hypoxic-ischemic early and long-term pathological lesions. All above demonstrated that Phenobarbital is neural protective against hypoxic-ischemic brain injure in neonatal rats.(2) Hypoxic-ischemic may have something to do with the down-regulation of KCC2mRNA and protein, while the treatment of Phenobarbital could increase the expression of KCC2.In conclusion, the treatment of Phenobarbital has neural protective effects in neonatal hypoxic-ischemic brain damage rats, one of the mechanisms might be prevent the down-regulation of KCC2. |
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