Supplementary Material for: Melatonin Reduces Hypoglycemia-Induced Neuronal Death in Rats

Melatonin, N-aceyl-5-methoxytryptamine, is the main secretory product of the pineal gland and has neuroprotective effects on several brain injuries, including ischemic stroke. In the present study, we hypothesized that exogenous melatonin may decrease hypoglycemia-induced neuronal death through the prevention of superoxide generation. To test our hypothesis, hypoglycemia was induced by injecting human insulin (10 U/kg, i.p.) in rats. Melatonin injection was started immediately after hypoglycemia (10 mg/kg, i.p.). The first melatonin injection was performed at the end of a 30-min isoelectric EEG period. The second and third injections were administered at 1 and 3 h after the first injection. Reactive oxygen species generation, as detected by dihydroethidium staining, was significantly reduced by melatonin treatment. Neuronal injury was reduced by the treatment of melatonin in the hippocampal CA1 and dentate granule cells. Microglia activation was robust in the hippocampus after hypoglycemia, which was almost completely prevented by melatonin treatment. Hypoglycemia-induced cognitive impairment was also significantly prevented by melatonin treatment. The present study suggests that melatonin has therapeutic potential to prevent hypoglycemia-induced brain injury.褪黑素（Melatonin，N-aceyl-5-methoxytryptamine）是松果体的主要分泌产物，对包括缺血性脑卒中在内的多种脑损伤具有神经保护活性。本研究提出假设：外源性褪黑素可通过抑制超氧阴离子生成，减轻低血糖诱导的神经元死亡。为验证该假说，我们通过向大鼠腹腔注射人胰岛素（10 U/kg，i.p.）构建低血糖模型。褪黑素干预于低血糖造模结束后即刻启动（10 mg/kg，i.p.）：首次注射于30分钟等电位脑电图周期结束时进行，第二次与第三次注射分别于首次注射后1小时和3小时完成。通过二氢乙啶染色检测发现，褪黑素治疗可显著降低活性氧生成。褪黑素治疗可减轻海马CA1区及齿状回颗粒细胞的神经元损伤。低血糖造模后海马区小胶质细胞激活显著，而褪黑素治疗可几乎完全阻断该激活过程。此外，褪黑素治疗还可显著改善低血糖诱导的认知功能损伤。本研究结果表明，褪黑素具备防治低血糖性脑损伤的治疗潜力。