Effect Of Diazepam On The Neonatal Rats With Hypoxia-ischaemia Brain Damage And Its Mechanism

Hypoxia-ischaemia brain damage (HIBD) is a major type of perinatal brain injuryin newborns and can often result in irreversible brain damage and a range ofneurological sequelae. Furthermore, it is a major cause of neonatal morbidity andmortality, especially for premature infants.Objective: In this study, we investigate the short-and long-term neuroprotectiveeffects of Diazepam on neonatal rats with HIBD and the potential mechanismsunderlying its protective effects. And provide a reference for the clinical treatment ofHIBD.Methods: Seven-day-old newborn Sprague-Dawley (SD) rats were randomly dividedinto sham group (Sham), hypoxia-ischaemia brain injury group (HIBD) anddiazepam-treatment group (Diazepam),20in each group. HIBD model is preparedaccording to the Rice-Vannucci method, Sham group were only free the left commoncarotid artery, and not expose to ischaemia and hypoxia. Diazepam was administratedimmediately via intraperitoneal (i.p.) injection after inducing HIBD at a dose of10mg.kg-1.8h-1for three consecutive days.The other two groups were given the samedose of saline solution.Three days after HIBD, rats were decapitated, and the extentof brain injury was evaluated using2,3,5-triphenyltetrazolium chloride (TTC)staining. Additionally, the expression of Potassium-chloride cotransporter-2(KCC2)was analysed using real-time PCR, Western blot analysis and immunohistochemistry.Three weeks after HIBD, rats were subjected to the Morris water maze test and thelocomotor activity test to determine the long-term therapeutic effects of Diazepam.Results:(1) Identification of the HIBD model: After hypoxia-ischaemia, neonatal ratsappear fixed to the left rotates, this indicating HIBD model was successfullyestablished.(2) TTC staining showed that: HIBD group has a large area of infarction area,once again shows the HIBD model was successful. The infarct ratio for the Diazepamgroup was noticeably lower compared to HIBD group. This indicates Diazepam has short-term neuroprotective effects on neonatal rats with HIBD.(3) Spontaneous activity showed: The locomotor activity for the HIBD group wassignificantly reduced compared with the Sham group (P<0.05). Rats were more activewhen given Diazepam, but there was no significant difference between HIBD group(P>0.05).(4) Morris water maze showed that5days total average escape latency inacquisition trials was: HIBD group>Diazepam group>Sham group. The probe trialshows that the sham group spent significantly more time in the target quadrant andhad more cross platform times when compared with the HIBD group. Improvementwas observed in the rats that were treated with Diazepam.(P<0.05or P<0.01).Theresults indicated that HIBD decreased ability of learning and memory, and earlyDiazepam treatment could improve long-term neurobehavioral.(5) KCC2mRAN and protein level were decreased significantly after the HIBD (P<0.05or P <0.01), there has some increase after Diazepam treatment.Conclusions:(1) Early Diazepam treatment can reduce the infarct ratio in HIBDnewborn-rats.This indicates Diazepam has short-term neuroprotective effects onneonatal rats with HIBD.(2) Early Diazepam treatment can improve the locomotor activity andlearning-memory function for HIBD newborn-rats. This indicates Diazepam haslong-term neuroprotective effects on neonatal rats with HIBD.(3) Diazepam plays a role in brain protection by preventing the decrease in KCC2expression.To sum up, early treatment with Diazepam appears to attenuate HIBD and canefficiently improve the long-term learning and memory capabilities of the animal. Apotential mechanism underlying these effects may involve preventing the decrease inKCC2expression, then maintain Cl-homoeostasis, ensuring GABA play a roleinhibitory neurotransmitter, and thus produce the neuroprotective effect.