Severe Global Cerebral Ischemia Induced Programmed Necrosis Of Hippocampal Neurons Is Prevented By 3-methyladenine, A Widely Used Inhibitor Of Autophagy

Severe global cerebral ischemia induced programmed necrosis in hippocampal neurons is prevented by 3-methyladenine, a widely used inhibitor of autophagyBackgroundIn clinical emergencies, many accidents lead to global ischemia and the brain is intrinsically more vulnerable to ischemia than other organs. Global cerebral ischemia results in selective neuronal death in the hippocampus and is called programmed cell death (PCD). Autophagic cell death is another type of PCD distinct from caspase-dependent apoptosis. There is also evidence that the extent of neuronal damage and the underlying mechanisms not only depend on the severity of the insult but also on the degree of brain maturation. To date, whether autophagy has beneficial or harmful effects in severe global cerebral ischemia/reperfusion (I/R) injury in adult animals is not well understood.ObjectivesIn this study we explored the features of neuronal death induced by 20-min severe global ischemia in adult Sprague-Dawley (SD) rats, and the role of apoptosis, autophagy and necrosis in this kind of neuronal death. Then we tested the role of 3-MA, a widely used autophagy inhibitor, in 20-min global cerebral I/R induced neuronal death.Methods1 Animals and surgical procedures:20 min transient global cerebral ischemia was induced by four-vessel occlusion (4-VO) technique in adult male rats.2 Five experiment groups:control, sham,600 nmol 3-MA was administered by intracerebroventricular injection (i.c.v.) at 60 min after reperfusion (R60) and at 30 min (130) or 60 min (160) before ischemia.3 Neuron Counts:5μm coronal sections at the level of the bregma were cut and stained with Hematoxylin/Eosin (HE). The number of surviving neurons in the hippocampal CA1 layer per 1 mm length was counted.4 Immunohistochemistry:The groups and coronal sections preparation in this study were the same with histological study. Cathepsin B and cleaved caspase-3 immunoreactivity was determined by the two-step methods immunohistochemistry. Negative and positive control was obtained at the same time.5 Deoxynucleotidyl transferase-mediated UTP nick end-labeling (TUNEL) staining:In situ labeling of DNA fragmentation was carried out with an in situ cell death detection kit followed the manufacturer's instructions.6 Transmission electron microscopy:The characteristic changes of neurons in hippocampal CA1 and DG areas were viewed under a transmission electron microscope.7 Western blot measurement:The total proteins of hippocampus were isolated, and the level of LC3Ⅰ/Ⅱ, caspase-3, cathepsin B was measured.Results1 The death of CA1 neurons induced by 20-min global ischemia occurred as a delayed manner, and 95.2% CA1 neurons was destroyed at 7 days of reperfusion.2 TUNEL-positive neurons increased markedly from 48 h to 72 h of reperfusion after 20-min global ischemia, which suggested that neuronal death was programmed.3 In EM study, no classical apoptotic morphology was observed in the CA1 and DG areas at any time of reperfusion. The number of autophagosomes (APs) and autolysosomes (ALs) in CA1 neurons after reperfusion increased greatly. The morphological changes of swollen organelles, broken organelle membranes, disrupted plasma membrane, as well as clumped chromatin seemed more close to the changes of necrosis.4 No significant expression and activation of caspase-3 were observed in the CA1 region from 12 h to 72 h of reperfusion after 20-min ischemia by Western blot and immunohistochemistry.5 The level of LC3-Ⅱincreased greatly from 1 h to 48 h of reperfusion after 20-min global ischemia, with a maximal induction at 12 h of reperfusion, which demonstrated the activation of autophagy.6 Twenty minutes global ischemia induced the release of cathepsin B from lysosomal into cytoplasm and nuclear after 24 h of reperfusion, but the expression and activation of cathepsin B had no significant change.7 Intracerebroventricular administration of 600 nmol 3-MA 30 min or 60 min before ischemia decreased CA1 pyramidal neuronal death significantly, the latter was more effective. While, the administration of 600 nmol 3-MA 60 min after reperfusion had no protective effect.8 The number of TUNEL-positive neurons were reduced nearly completely by 3-MA administered 60 min before ischemia, and partly by 3-MA administered 30 min before ischemia, however administration of 3-MA 60 min after reperfusion has no such effect.9 The release of cathepsin B was inhibited greatly by 3-MA administered 60 min before ischemia, and partly by 3-MA administered 30 min before ischemia; however the administration 60 min after reperfusion has no significant effect.Conclusions1 The neuronal death in hippocampal CA1 area induced by 20-min global I/R injury was more likely to be "programmed necrosis", which might be induced by the release of cathepsin B, a lysosomal enzyme.2 600 nmol 3-MA had time dependent protective effect on this kind of neuronal death. The protective effect of 3-MA might be not only through autophagy inhibition, but also through inhibition the release of lysosomal enzyme, the latter might be more critical.3 Our study demonstrated another important protective mechanism of 3-MA for the first time:protected against "programmed necrosis". The interactions among autophagy, apoptosis, and necrosis are complex with much cross-talk, and their role in ischemia remains worth further study to find effective therapeutic strategies for repair of ischemic cerebrovascular injury.