Autophagy Is Involved In Hypoxic Postconditioning-induced Neuroprotection Against Transient Global Cerebral Ischemia In Adult Rats

Background and Purpose: Transient global cerebral ischemia inducesdelayed death of neurons selectively in hippocampus. Ischemic toleranceis an adaptive defense in which brain exposes to noninjurious damageand can alleviate subsequently severe lethal injury. The methods ofischemic tolerance include noninjurious ischemia, brief episodes ofseizure, low doses of endotoxin, cortical spreading depression, andexposure to anesthetic inhalants. The mechanisms of neuroprotectioninduced by hypoxic postconditioning are unclear. Our previous studyshowed that TUNEL-positive cells in the hippocampal CA1subregion oftGCI groups were increased24h after reperfusion, and stronglyincreased at48h. It is not clear whether autophagy participates intGCI-induced damage and HPC-induced neuroprotection. In the presentstudy, immunohistochemistry and immunoblotting were used to detectLC3, LAMP2and Cathepsin D in hippocampal CA1subregion after tGCIand HPC. Transmission electron microscopy was used to detectautophagosome formation. The present study aims to explore whetherautophagy participates in neuroprotection induced by HPC. Material and methods. Adult male Wistar rats weighing250-300g(Southern Medical University, Guangdong, China) were used in our study.tGCI was induced by applying the four-vessel occlusion method. Hypoxiapostconditioning was performed by expositing rats to a2h period ofsystemic hypoxia (8%O2+92%N2)24h after a tGCI. TEM was used todetect ultrastructure of neurons and autophagosome formation.Immunohistochemistry and immunoblotting were used to detect LC3,LAMP2and Cathepsin D in hippocampal CA1subregion after tGCI withor without HPC.Results:(1) TEM demonstrated that neurons in the sham groupappeared to be normal with healthy-looking endoplasmic reticulum, nuclei,mitochondria. Neurons in hippocampal CA1subregion demonstratedswollen mitochondria and sporadic autophagosome26h after tGCI. Also,autophagosome increased obviously50h after tGCI. Neurons showeddestructive cell structure and necrosis7d after tGCI. However, neuronsin hippocampal CA1subregion of HPC appeared relatively intactorganelles including mitochondria, nuclei, lysosome and severalautophagosomes.(2) Immunohistochemistry showed that LC3labelingwas diffuse and evenly distributed in the cytosol of hippocampal neurons.After tGCI, LC3gathered near cell membrane and HPC alleviated theredistribution. Western blot showed that LC3-Ⅱ/Ⅰ increased graduallyand peaked at50h after tGCI.(3) LAMP2-immunoreactive cells in thepyramidal cell layers were round or ovoid (neuron-like). Immunoreactivecells in molecular layer and polymorph layer were spindle or stellated(astrocyte-like). Neuron-like cells in hypoxia without ischemia groupevidently increased24h after hypoxia. Also, the number of neuron-likecells increased transiently4h and decreased at168h.Immediately after hypoxia, HPC was lower than tGCI. Compared withsham, there were no significantly changes in the astrocyte-like cells afterhypoxia. However, the astrocyte-like cells increased immediately after tGCI and peaked at50h. In the HPC group, the astrocyte-like cellsfurther increased6days after hypoxia compared with the correspondingtGCI group. As shown in Western blot analysis, LAMP2significantlyincreased24hours after hypoxia. In the tGCI groups, LAMP2showed nosignificant changes except for an increase immediately after tGCI. In theHPC groups, no significant changes were observed when comparing withthe corresponding tGCI groups.(4) The soma of Cathepsin Dimmunoreactive cell was big and round, whereas the dendrites inmolecular layer were long and obvious in hippocampal CA1subregion inthe sham-operated group. Compared with sham-operated group, nosignificant changes in the optical density of Cathepsin D were observed inrats with hypoxia. The immunostaining of Cathepsin D was significantlydecreased at0h,50h and168h after tGCI. After HPC, immunostainingof Cathepsin D recovered to the level of sham group. Western blotrevealed that level of Cathepsin D increased24h after hypoxia. After tGCI,Cathepsin D increased at0-26h and decreased to the level of sham at50h after tGCI. In the HPC group, no significant changes were observed at0h and24h after hypoxia compared with the corresponding tGCI groups.(5) Immunofluorescent double staining showed that LAMP2waspredominantly expressed in the microglia of CA1in sham groups. In theventromedial region of CA1, LAMP2was expressed in the cytoplasm ofneurons. However, LAMP2was almost completely expressed in themicroglia7d after tGCI.Conclusion:(1) Autophagy was activated after tGCI, which was probablyrelated to neuronal death.(2) Autophagy was activated after HPC andlysosome associated protein increased to digest autophagosome. Thismaybe related to neuroprotection.(3) Activation of lysosome in themicroglia probably participates in the neuroprotection of HPC againsttGCI.