KCC2 expression changes in Diazepam-treated neonatal rats with hypoxia-ischaemia brain damage

• This is the first report of changes in the KCC2 expression level in a Diazepam HIBD in vivo model.
• Short- and long-term neuroprotective effects of Diazepam were first described in an HIBD model.
• The regulation of KCC2 expression is a possible mechanism the neuroprotective effect of Diazepam.

Hypoxia-ischaemia brain damage (HIBD) is a major type of perinatal brain injury in newborns. In this study, we investigate the short- and long-term neuroprotective effects of Diazepam on neonatal rats with HIBD and the potential mechanisms underlying its protective effects. Seven-day-old Sprague-Dawley rats were subjected to left carotid artery ligation followed by a 2-h exposure to 8% oxygen and 92% nitrogen. Diazepam was administered immediately via intraperitoneal (i.p.) injection after inducing HIBD at a dose of 10 mg kg−1 8 h−1 for three consecutive days. Three days after HIBD, rats were decapitated, and the extent of brain injury was evaluated using 2,3,5-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 (MWM) test and the locomotor activity test to determine the long-term therapeutic effects of Diazepam. We observed that the volume of infarction in the Diazepam group was significantly less (P<0.01) compared with the HIBD group. We also observed that the learning and memory abilities of the Diazepam rats improved significantly compared with the untreated rats (P<0.05) and that the decrease in KCC2 expression was prevented (P<0.01). Early treatment with Diazepam appears to attenuate HIBD and can efficiently improve the long-term learning and memory capabilities of the animal. A potential mechanism underlying these effects may involve preventing the decrease in KCC2 expression.