Objective To investigate the rat model establishment of repetitive and profound hypoglycemia of neonatal period (RPHN),and understand related brain injury and its prognosis.Methods Twenty-four 2 day-old Sprague-Dawley rats were randomly divided into four groups with six in each:the insulin-treated neonatal rats group (INS-N),control neonatal rats group (CON-N),insulin-treated neonatal rats followed up to adolescent stage group (INS-A) and control neonatal rats followed up to adolescent stage group (CON-A).Rats in INS N group and INS-A group received intraperitoneal injections of insulin (40 U/kg) on postnatal day 2,4 and 6 to induce severe hypoglycemia (≤ 1.4 mmol/L).The rats in corresponding control group received the same amount of saline.Terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay (TUNEL) was applied to study the amounts of injured neurons in occipital cortex and hippocampus of the rats in their neonatal period and adolescent period.The difference between the two groups was compared by t test.Results In INS-N and INS-A group,neonatal rats reached severe hypoglycemia level at 2.5 h after insulin injection (glucose at day 2＜1.3 mmol/L,at day 4 and day 6＜1.4 mmol/L).RPHN was observed to induce neuron injury in hippocampus CA1,CA3,DG,and occipital cortex in the INS N group (numbers of neuron injury in INS-N group:23±5,28±5,234±26 and 187± 17,respectively; in CON N group:14±3,16±3,26±4 and 30±6) (t=2.97,3.11,4.08 and 3.52,all P＜0.05).Compared with CON-N group,INS N group had higher injured index in above brain regions (INS-N group:0.22±0.04,0.24±0.06,0.83±0.06 and 0.34±0.08,respectively; CON-N group:0.12±0.02,0.13±0.02,0.16±0.03 and 0.17±0.04) (t=3.42,3.68,3.92 and 2.76,all P＜0.05).When the newborn rats reached their adolescent stage,there were more injured neurons in INS-A group in above brain regions (INS-A group:54± 7,42±9,296±34 and 75± 12,respectively; CON-A group:19±3,16±2,31 ±5 and 20±4) (t=3.47,3.24,3.80 and 3.92,all P＜0.05),and also showed higher injured index (INS-A group:0.86±0.12,0.89±0.14,0.92±0.09and 0.54±0.11,respectively; CON-A group:0.14±0.03,0.13±0.02,0.17±0.02 and 0.15±0.04) (t=3.05,3.60,3.52 and 3.33,all P＜0.05).Conclusion RPHN rat model can be produced by intraperitoneal injection of insulin (40 U/kg) on day 2,4 and 6.RPHN causes neuron injury in the occipital cortex and hippocampus,and such injury may last to adolescent stage.