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Subcortical ischemic vascular dementia is caused by chromic ischemia due to narrowing of small blood vessels supplying the white matter.The pathological changes of ischemic white matter degeneration include axonal degeneration,demyelination,and glial activation.However,it is not fully understood how chronic ischemia leads to the white matter pathologies.In this study,we aimed to develop an in vitro model of ischemic white matter degeneration using cultured cerebellar slices.Cere-bellar slices were obtained from postnatal day 12 mice and cultured for 12 days in vitro.At this time point,subcortical white matter axon bundles survived with exuberant myelination.Most of surviving axons from cerebellar cortices were positive for Purkinje cell marker calbindin-D28k.The cultured cerebellar slices were exposed to 2% hypoxic condition for 48 hours beginning at 10 days in vitro.Hypoxic insult resulted in a beaded appearance of the axons characterized by focal swelling and constriction.In contrast,myelin basic protein expression was preserved even at the site of severe axonal damages.Moreover,there was no significant decrease or loss of oligodendrocytes,suggesting that axonal structures are more vulnerable to hypox-ia than myelin ensheathment.Electron micrographs showed accumulation of lysosome-like structures in the axoplasm after hypoxia.Consistent with the electron microscopic findings,lysosomal-associated membrane protein 1 (LAMP1) immune-reactivity was significantly increased in the white matter under hypoxic conditions.The AMPA/kainate receptor antagonist NBQX,calpain inhibitor ALLN and MDL28170,and lysosomal protease inhibitor pepstatin A,significantly attenuated hy-poxia-induced beading appearances of the axons.Our slice models could be utilized to explore molecular mechanisms of ischemia-induced axon degeneration.Axonal degeneration following hypoxia is likely to involve multiple pathways,such as AMPA receptor,calpain proteolysis and lysosomal proteases.